Methods for producing enucleated erythroid cells derived from pluripotent stem cells

ABSTRACT

Methods for generating enucleated erythroid cells using pluripotent stem cells are provided. The methods permit the production of large numbers of cells. The cells obtained by the methods disclosed may be used for a variety of research, clinical, and therapeutic applications. Methods for generating megakaryocyte and platelets are also provided.

FIELD OF INVENTION

The present invention relates to producing human enucleated erythroidcells from pluripotent stem cells.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

There is a critical need for available blood for transfusion. The RedCross and other suppliers of blood report a near constant shortage ofblood. This is especially true for patients with unique blood types,patients who are Rh+, or following accidents or disasters resulting inmass casualties. Additionally, in times of war, the military has anacute need for available blood for use in the treatment of traumaticwar-related injuries. The present invention provides improved methodsand compositions for use in blood banking and transfusion. The cells andmethods of the present invention will provide a safe and reliableadvance beyond the traditional reliance on blood donations, and willhelp prevent critical shortages in available blood.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described andillustrated in conjunction with compositions and methods which are meantto be exemplary and illustrative, not limiting in scope.

The present invention provides methods for making and using erythroidcells and enucleated erythroid cells derived from pluripotent stemcells.

In certain embodiments, the present invention provides for a method ofproducing a pluripotent stem cell-derived enucleated erythroid cell,comprising: providing a pluripotent stem cell; and differentiating saidpluripotent stem cell into an enucleated erythroid cell by culturingsaid pluripotent stem cell with OP9 mouse stromal cells or humanmesenchymal stem cells (MSCs).

In certain embodiments, differentiating said pluripotent stem cell intoan enucleated erythroid cell comprises differentiating said pluripotentstem cell into a hemangioblast, non-engrafting hemangio cell or blastcell. In certain embodiments, said hemangioblast, non-engraftinghemangio cell, or blast cell is expanded prior to being differentiatedinto said enucleated erythroid cell. In certain embodiments, saidhemangioblasts, non-engrafting hemangio cells, or blast cells areexpanded in Stemline II medium with Epo, IL-3, and SCF.

In certain embodiments, said pluripotent stem cell is a humanpluripotent stem cell and differentiating said human pluripotent stemcell into said hemangioblast is done in vitro by a method comprising:(a) culturing a cell culture comprising human pluripotent stem cell inserum-free media in the presence of at least one growth factor in anamount sufficient to induce the differentiation of said humanpluripotent stem cell into embryoid bodies; and (b) adding at least twogrowth factors to said culture comprising embryoid bodies and continuingto culture said culture in serum-free media, wherein said growth factoris in an amount sufficient to expand said human hemangioblast in saidembryoid bodies culture, wherein said human pluripotent stem cells,embryoid bodies and hemangioblasts are grown in serum-free mediathroughout steps (a) and (b) of said method, and wherein said at leasttwo growth factors in step (b) comprise BMP4 and VEGF. In certainembodiments, differentiating said human pluripotent stem cell into saidhemangioblast further comprises (c) disaggregating said embryoid bodiesinto single cells; and (d) adding at least one growth factor to saidculture comprising said single cells and continuing to culture saidculture in serum-free media, wherein said growth factor is in an amountsufficient to expand human hemangioblasts in said culture comprisingsaid single cells, and wherein said human pluripotent stem cells,embryoid bodies and hemangio-colony forming cells are grown inserum-free media throughout steps (a)-(d) of said method.

In certain embodiments, said pluripotent stem cell is a humanpluripotent stem cell and differentiating said human pluripotent stemcell into said non-engrafting hemangio cell is done in vitro by a methodcomprising: (a) culturing a cell culture comprising said humanpluripotent stem cell in serum-free media in the presence of at leastone growth factor in an amount sufficient to induce the differentiationof said human pluripotent stem cell into embryoid bodies; and (b) addingat least one growth factor to said culture comprising embryoid bodiesand continuing to culture said culture in serum-free media, wherein saidgrowth factor is in an amount sufficient to expand said humannon-engrafting hemangio cell in said embryoid bodies culture, whereinsaid embryoid bodies are cultured for 10-13 days, and wherein said humanpluripotent stem cell, embryoid bodies and non-engrafting hemangio cellsare grown in serum-free media throughout steps (a) and (b) of saidmethod. In certain embodiments, differentiating said pluripotent stemcell into said non-engrafting hemangio cell further comprises (c)disaggregating said embryoid bodies into single cells; and (d) adding atleast one growth factor to said culture comprising said single cells andcontinuing to culture said culture in serum-free media, wherein saidgrowth factor is in an amount sufficient to expand said humannon-engrafting hemangio cell in said culture comprising said singlecells, wherein said embryo-derived cells, embryoid bodies andnon-engrafting hemangio cells are grown in serum-free media throughoutsteps (a)-(d) of said method.

In certain embodiments, differentiating said pluripotent stem cell intosaid enucleated erythroid cell further comprises culturing saidpluripotent stem cell in the culture medium comprising EPO. In certainembodiments, differentiating said pluripotent stem cell into saidenucleated erythroid cell further comprises: culturing said pluripotentstem cell in a culture medium comprising a supplement selected from thegroup consisting of inositol, folic acid, monothioglycerol, transferrin,insulin, ferrous nitrate, ferrous sulfate, BSA, L-glutamine,penicillin-streptomycin and combinations thereof; and culturing saidpluripotent stem cell in said culture medium wherein said culture mediumfurther comprises an agent selected from the group consisting ofhydrocortisone, SCF, IL3, Epo and combinations thereof.

In certain embodiments, said pluripotent stem cell used in the presentinvention is an embryonic stem cell or embryo-derived cell. In certainembodiments, said pluripotent stem cell is an induced pluripotent stemcell. In certain embodiments, said pluripotent stem cell is a humancell. In certain embodiments, said pluripotent stem cell is geneticallymanipulated prior to differentiation.

In certain embodiments, said growth factor used in the present inventionis a fusion protein that comprises HOXB4 and a protein transductiondomain (PTD). In certain embodiments, said HOXB4 is mammalian HOXB4. Incertain embodiments, said mammalian HOXB4 is mouse or human HOXB4.

In certain embodiments, said growth factor used in the present inventionis selected from the group consisting of vascular endothelial growthfactor (VEGF), bone morphogenic proteins (BMP), stem cell factor (SCF),Flt-3L (FL) thrombopoietin (TPO) and erythropoietin (EPO). In certainembodiments, said vascular endothelial growth factor (VEGF), bonemorphogenic protein (BMP), or both, are added to step (a) within 0-48hours of cell culture. In certain embodiments, said stem cell factor(SCF), Flt-3L (FL) or thrombopoietin (TPO), or any combination thereof,are added to said culture within 48-72 hours from the start of step (a).

In certain embodiments, the methods further comprise the step of addingerythropoietin (EPO) to step (a) or further comprises the step of addingerythropoietin (EPO) to step (a) or (d).

In certain embodiments, the present invention provides enucleatederythroid cells produced by methods as described above.

Other embodiments of the present invention also provides a method ofproducing a pluripotent stem cell-derived erythroid cell, comprising:providing a pluripotent stem cell; and differentiating said pluripotentstem cell into an erythroid cell by culturing said pluripotent stem cellin a medium comprising EPO.

In certain embodiments, differentiating said pluripotent stem cell intoan erythroid cell comprises differentiating said pluripotent stem cellinto a hemangioblast, non-engrafting hemangio cell, or blast cell. Incertain embodiments, said hemangioblast, non-engrafting hemangio cell,or blast cell is expanded prior being differentiated into said erythroidcell. In certain embodiments, said hemangioblasts, non-engraftinghemangio cells, or blast cells are expanded in Stemline II medium withEpo, IL-3, and SCF.

In certain embodiments, said pluripotent stem cell is a humanpluripotent stem cell and differentiating said human pluripotent stemcell into said hemangioblast is done in vitro by a method comprising:(a) culturing a cell culture comprising said human pluripotent stem cellin serum-free media in the presence of at least one growth factor in anamount sufficient to induce the differentiation of said humanpluripotent stem cell into embryoid bodies; and (b) adding at least twogrowth factors to said culture comprising embryoid bodies and continuingto culture said culture in serum free media, wherein said growth factoris in an amount sufficient to expand said human hemangioblast in saidembryoid bodies culture, wherein said human pluripotent stem cells,embryoid bodies and hemangioblasts are grown in serum-free mediathroughout steps (a) and (b) of said method, and wherein said at leasttwo growth factors in step (b) comprise BMP4 and VEGF.

In certain embodiments, differentiating said human pluripotent stem cellinto said hemangioblast further comprises (c) disaggregating saidembryoid bodies into single cells; and (d) adding at least one growthfactor to said culture comprising said single cells and continuing toculture said culture in serum-free media, wherein said growth factor isin an amount sufficient to expand human hemangioblasts in said culturecomprising said single cells, and wherein said pluripotent stem cells,embryoid bodies and hemangio-colony forming cells are grown inserum-free media throughout steps (a)-(d) of said method.

In certain embodiments, said pluripotent stem cell is a humanpluripotent stem cell and differentiating said pluripotent stem cellinto said non-engrafting hemangio cell is done in vitro by a methodcomprising: (a) culturing a cell culture comprising human pluripotentstem cell in serum-free media in the presence of at least one growthfactor in an amount sufficient to induce the differentiation of saidhuman pluripotent stem cell into embryoid bodies; and (b) adding atleast one growth factor to said culture comprising embryoid bodies andcontinuing to culture said culture in serum-free media, wherein saidgrowth factor is in an amount sufficient to expand said humannon-engrafting hemangio cells in said embryoid bodies culture, whereinsaid embryoid bodies are cultured for 10-13 days, and wherein said humanpluripotent stem cell, embryoid bodies and non-engrafting hemangio cellsare grown in serum-free media throughout steps (a) and (b) of saidmethod.

In certain embodiments, differentiating said pluripotent stem cell intosaid non-engrafting hemangio cell further comprises (c) disaggregatingsaid embryoid bodies into single cells; and (d) adding at least onegrowth factor to said culture comprising said single cells andcontinuing to culture said culture in serum-free media, wherein saidgrowth factor is in an amount sufficient to expand human non-engraftinghemangio cells in said culture comprising said single cells, whereinsaid human pluripotent stem cell, embryoid bodies and non-engraftinghemangio cells are grown in serum-free media throughout steps (a)-(d) ofsaid method.

In certain embodiments, said pluripotent stem cell used in the presentinvention is an embryonic stem cell or embryo-derived cell. In certainembodiments, said pluripotent stem cell is an induced pluripotent stemcell. In certain embodiments, said pluripotent stem cell is a humancell. In certain embodiments, said pluripotent stem cell is geneticallymanipulated prior to differentiation.

In certain embodiments, said growth factor used in the present inventionis a fusion protein that comprises HOXB4 and a protein transductiondomain (PTD). In certain embodiments, said HOXB4 is mammalian HOXB4. Incertain embodiments, said mammalian HOXB4 is mouse or human HOXB4.

In certain embodiments, said growth factor used in the present inventionis selected from the group consisting of vascular endothelial growthfactor (VEGF), bone morphogenic proteins (BMP), stem cell factor (SCF),Flt-3L (FL) thrombopoietin (TPO) and erythropoietin (EPO). In certainembodiments, said vascular endothelial growth factor (VEGF), bonemorphogenic protein (BMP), or both, are added to step (a) within 0-48hours of cell culture. In certain embodiments, said stem cell factor(SCF), Flt-3L (FL) or thrombopoietin (TPO), or any combination thereof,are added to said culture within 48-72 hours from the start of step (a).

In certain embodiments, the methods further comprise the step of addingerythropoietin (EPO) to step (a) or further comprises the step of addingerythropoietin (EPO) to step (a) or (d).

In certain embodiments, the present invention provides erythroid cellsproduced by methods as described above.

Still other embodiments of the present invention provides methods ofproducing a megakaryocyte or a platelet, comprising: providing apluripotent stem cell; differentiating said pluripotent stem cell into ahemangioblast, non-engrafting hemangio cell, or blast cell; anddifferentiating said hemangioblast, non-engrafting hemangio cell, orblast cell into said megakaryocyte or said platelet by culturing inmegakaryocyte (MK) culture medium comprising TPO.

In certain embodiments, said pluripotent stem cell used in the presentinvention is an embryonic stem cell or embryo-derived cell. In certainembodiments, said pluripotent stem cell is an induced pluripotent stemcell. in certain embodiments, said pluripotent stem cell is a humancell. In certain embodiments, said pluripotent stem cell is geneticallymanipulated prior to differentiation. In certain embodiments, saidhemangioblast, non-engrafting hemangio cell, or blast cell is expandedprior to being differentiated into said megakaryocyte or said platelet.

In certain embodiments, said hemangioblasts, non-engrafting hemangiocells, or blast cells are expanded in Stemline II medium with Epo, IL-3,and SCF.

In certain embodiments, said pluripotent stem cell is a humanpluripotent stem cell and differentiating said human pluripotent stemcell into said hemangioblast is done in vitro by a method comprising:(a) culturing a cell culture comprising human pluripotent stem cell inserum-free media in the presence of at least one growth factor in anamount sufficient to induce the differentiation of said humanpluripotent stem cell into embryoid bodies; and (b) adding at least twogrowth factors to said culture comprising embryoid bodies and continuingto culture said culture in serum-free media, wherein said growth factoris in an amount sufficient to expand said human hemangioblast in saidembryoid bodies culture, wherein said human pluripotent stem cells,embryoid bodies and hemangioblasts are grown in serum-free mediathroughout steps (a) and (b) of said method, and wherein said at leasttwo growth factors in step (b) comprise BMP4 and VEGF.

In certain embodiments, differentiating said human pluripotent stem cellinto said hemangioblast further comprises: (c) disaggregating saidembryoid bodies into single cells; and (d) adding at least one growthfactor to said culture comprising said single cells and continuing toculture said culture in serum-free media, wherein said growth factor isin an amount sufficient to expand human hemangioblasts in said culturecomprising said single cells, and wherein said human pluripotent stemcells, embryoid bodies and hemangio-colony forming cells are grown inserum free media throughout steps (a)-(d) of said method.

In certain embodiments, differentiating said hemangioblast,non-engrafting hemangio cell, or blast cell into said megakaryocyte orsaid platelet is done after about 6 to 8 days of hemangioblast,non-engrafting hemangio cell, or blast cell culture.

In certain embodiments, said pluripotent stem cell is a humanpluripotent stem cell and differentiating said human pluripotent stemcell into said non-engrafting hemangio cell is done in vitro by a methodcomprising: (a) culturing a cell culture comprising said humanpluripotent stem cell in serum-free media in the presence of at leastone growth factor in an amount sufficient to induce the differentiationof said human pluripotent stem cell into embryoid bodies; and (b) addingat least one growth factor to said culture comprising embryoid bodiesand continuing to culture said culture in serum-free media, wherein saidgrowth factor is in an amount sufficient to expand said humannon-engrafting hemangio cell in said embryoid bodies culture, whereinsaid embryoid bodies are cultured for 10-13 days, and wherein said humanpluripotent stem cell, embryoid bodies and non-engrafting hemangio cellsare grown in serum-free media throughout steps (a) and (b) of saidmethod.

In certain embodiments, differentiating said pluripotent stem cell intosaid non-engrafting hemangio cell further comprises: (c) disaggregatingsaid embryoid bodies into single cells; and (d) adding at least onegrowth factor to said culture comprising said single cells andcontinuing to culture said culture in serum free media, wherein saidgrowth factor is in an amount sufficient to expand said humannon-engrafting hemangio cell in said culture comprising said singlecells, wherein said embryo-derived cells, embryoid bodies andnon-engrafting hemangio cells are grown in serum-free media throughoutsteps (a)-(d) of said method.

In certain embodiments, said growth factor used in the present inventionis a fusion protein that comprises HOXB4 and a protein transductiondomain (PTD). In certain embodiments, said HOXB4 is mammalian HOXB4. Incertain embodiments, said mammalian HOXB4 is mouse or human HOXB4.

In certain embodiments, said growth factor used in the present inventionis selected from the group consisting of vascular endothelial growthfactor (VEGF), bone morphogenic proteins (BMP), stem cell factor (SCF),Flt-3L (FL) thrombopoietin (TPO) and erythropoietin (EPO). In certainembodiments, said vascular endothelial growth factor (VEGF), bonemorphogenic protein (BMP), or both, are added to step (a) within 0-48hours of cell culture. In certain embodiments, said stem cell factor(SCF), Flt-3L (FL) or thrombopoietin (TPO), or any combination thereof,are added to said culture within 48-72 hours from the start of step (a).

The present invention also provides a megakaryocyte or a plateletproduced by any one of the method as described above.

Still other embodiments, the invention provides a method of producing anenucleated erythroid cell comprising the steps of (a) providing apluripotent stem cell; and (b) differentiating said pluripotent stemcell into enucleated erythroid cells. In certain embodiments, saidpluripotent stem cell is an embryonic stem cell or embryo-derived cell.In certain embodiments, said pluripotent stem cell is an inducedpluripotent stem cell. In certain embodiments, said pluripotent stemcell is a human cell. In certain embodiments, said pluripotent stem cellis genetically manipulated prior to differentiation. In certainembodiments, said pluripotent stem cell is differentiated intohemangioblasts (e.g., hemangioblasts, hemangio colony forming cells,hemangio cells, non-engrafting hemangio cells, or blast cells) prior tostep (b). In certain embodiments, said hemangioblasts or blast cells areexpanded prior to step (b). In certain embodiments, hemangioblasts,non-engrafting hemangio cells, or blast cells are expanded about 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 days. In certain embodiments,hemangioblasts, non-engrafting hemangio cells, or blast cells areexpanded from about day 3.5 to about day 10. In certain embodiments,said hemangioblasts, non-engrafting hemangio cells, or blast cells areexpanded in Stemline II medium with Epo, IL-3, and SCF. In certainembodiments, hemangioblasts or blast cells are differentiated for about5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days. In certain embodiments,hemangioblasts, non-engrafting hemangio cells, or blast cells aredifferentiated from about day 11 to about day 20. In certainembodiments, said enucleated erythroid cells are cultured with OP9 orMSC cells. In certain embodiments, said culture is supplemented withEpo. The invention contemplates all suitable combinations of any of theforgoing or following aspects and embodiments of the invention.

In certain embodiments, the present invention provides enucleatederythroid cells produced by methods as described above.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousfeatures of embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 depicts large scale production of erythroid cells from hESCs inaccordance with an embodiment of the present invention. (A) Erythroidcells (pellet) derived from 2×10⁶ human ESCs. (B), erythroid cells fromFIG. 1A were resuspended in equivalent hematocrit of human whole blood;(C, D) Morphology of erythroid cells derived from human ESCs (C,(originally 200× and D, originally 1000×). (E) Electrospray ionizationmass spectra of globin chains in hemoglobins from hESC-derived erythroidcells, confirming the presence of α, ζ, ε and Gγ globins. The observedmolecular weight for each of the globins is shown. (F) Flow cytometryanalysis of hESC-derived erythroid cells. Erythroid cells derived fromhESCs were labeled with specific antibodies conjugated with PE andanalyzed on a FacScan flow cytometer (Becton Dickinson) with theCellQuest program. Corresponding unspecific isotype antibodiesconjugated with the same dyes were used as negative controls.

FIG. 2 depicts functional characterization of hESC-derived erythroidcells in accordance with an embodiment of the present invention. (A)Oxygen equilibrium curves of normal human RBCs and human ESC-derivederythroid cells. Note, the two curves are virtually indistinguishable attheir midpoints, whereas the curve of human ESC-derived erythroid cellsis leftward shifted at low (arrow) and high (arrow head) oxygensaturation percentages. (B) The Bohr effect. (C) Effects of 2,3-DPGdepletion. The solid lines represent the normal RBC control and thedashed lines represent the human ESC-derived erythroid cells. For eachpair, the line on the right represents the fresh cells and the one tothe left is the curve from cells depleted of 2,3-DPG.

FIG. 3 depicts characterization of Rh(D) and ABO genotype of hESC linesby PCR in accordance with an embodiment of the present invention. (A)Genotyping of RhD locus: Specific primers were designed for the Rh locusthat when Rh(D) positive DNA was used, 1,200-bp (weak) and 600-bp PCRproducts were amplified; whereas DNA from RhD-negative cells generatedonly the 1,200-bp fragment. (B, C) Genotyping of the ABO locus: twopairs of primers were designed to amplify two regions of the ABO locus.The PCR products were digested with restriction enzymes to distinguishABO types. ABO and Rh(D) genotypes are as follows: WA01, O(+); MA99,B(−); MA133, A(−); WA07 and MA09, B(+); and WA09 and MA01, A(+). (D) RhDantigen expression analysis on erythroid cells derived from MA01 andMA99 hESCs by FACS. Erythroid cells generated from MA01 and MA99 hESCswere stained with PE-labeled monoclonal anti-RhD antibody and analyzedby FACS. (E) ABO type characterization of hESC-derived erythroid cells.Panel A (originally 400×), cells stained with monoclonal antibodyagainst A-antigen; Panel B (originally 400×), cells stained withmonoclonal antibody against B-antigen.

FIG. 4 depicts enucleation of hESC-derived erythroid cells in vitro inaccordance with an embodiment of the present invention. (A) Diameterdecreases with time in culture. Data for each day represent diameters ofnucleated cells except “27e” represents diameters of enucleated cells at27 days. Enucleated cells decrease to less than half the originaldiameter on day 8. (B) Nuclear to cytoplasm ratio decreases with time inculture. Samples significantly different from day 8 are denoted by*=P<0.05,**=P<0.001, #=P<0.002. (C, E) Erythroid cells derived fromhuman ESCs were cultured in vitro for four weeks in Stemline II mediawith supplements and co-cultured with OP9 stromal cells on day 36. Onday 42, cells were cytospun and stained with Wright-Giemsa dye. (C,originally 200× and E, originally 1000×); (D, F) Red blood cells fromhuman blood were also cytospun and stained with Wright-Giemsa andcompared with hESC-derived erythroid cells. (D, originally 200× and F,originally 1000×) Scale bar=10 μm.

FIG. 5 depicts maturation of hESC-derived erythroid cells mimicerythroid development in accordance with an embodiment of the presentinvention. (A) Expression of CD235a, a mature erythrocyte marker,increases with time and CD71, an immature red blood cell marker, shows adecrease in expression over time. (B) Expression of β-globin chain inhESC-derived erythroid cells. Cytospin samples of hESC-derived erythroidcells collected from day 17 and day 28 differentiation and maturationcultures were stained with human β-globin chain specific antibody. (C)Progressive maturation of hESC-derived erythroid cells in vitro.Progressive morphological changes from blast cells to erythroblasts, andeventually matured erythrocytes are accompanied by significant increaseof hemoglobin and decrease in size during their in vitro differentiationand maturation. Cells were stained with both Wright-Giemsa and benzidine(A and B, originally 200×).

FIG. 6 depicts expression of glyphorin A in hESC-derived erythroid cellsin accordance with an embodiment of the present invention. Cytospinsamples of hESC-derived erythroid cells collected from day 28differentiation and maturation cultures were stained with human CD235aantibody. Almost 100% of cells stained positive for CD235a. (originally200×).

FIG. 7 depicts expression of β-globin chain in hESC-derived erythroidcells in accordance with an embodiment of the present invention.Cytospin samples of hESC-derived erythroid cells collected from day 28differentiation and maturation cultures were stained with human β-globinchain specific antibody. (originally 200×).

FIG. 8 depicts analysis of β-cluster globin gene expressions by RT-PCRin accordance with an embodiment of the present invention. Erythroidcells differentiated at different stages were collected and theexpression of β-, γ- and ε-globin genes was analyzed by RT-PCR usingglobin chain specific primers. RNA from adult bone marrow cells was usedas a positive control for β-globin gene and a negative control forε-globin gene. Day 28a and Day 28b are erythroid cells from two separateexperiments. BM, bone marrow.

FIG. 9 depicts the effects of BMPs and VEGF₁₆₅ on the development ofblast colonies in accordance with an embodiment of the presentinvention. A. Different doses of BMP-4 were added in EB mediumcontaining 50 ng/ml of VEGF₁₆₅, and a dose dependent development ofblast colonies was observed for BMP-4. B. EB medium containing 50 ng/mlof BMP-4 and VEGF₁₆₅ were supplemented with different doses (0, 10 and20 ng/ml) of BMP-2 and BMP-7. BMP-2 and BMP-7 failed in promoting blastcolony development. C. Different doses of VEGF₁₆₅ were added in EBmedium containing 50 ng/ml of BMP-4. The development of blast coloniesis VEGF₁₆₅ dose dependent. **P<0.01, n=3. 1×10⁵ cells from day 3.5 EBswere plated per well.

FIG. 10 depicts the effect of bFGF on the development of blast coloniesadded during different stages in accordance with an embodiment of thepresent invention. (a) Different doses of bFGF were added in EB medium;(b) Different doses of bFGF were supplemented in blast colony growthmedium (BGM); (c) Different doses of bFGF were added in both EB mediumand BGM. **P<0.01, n=3. B and C. Net-work like structure formation ofendothelial cells derived from BCs developed in BGM with (B) and without(C) bFGF. Endothelial cells from both sources formed net-work likestructures with no obvious difference.

FIG. 11 depicts the effect of bFGF on the development of blast coloniesfrom three hESC lines in accordance with an embodiment of the presentinvention. Diagonal Strips Different doses of bFGF were added in BGM.Horizontal Strips: Various doses of bFGF were added in EB medium.*P<0.05; **P<0.01, n=3.

FIG. 12 depicts hESC grown under feeder-free conditions retainpluripotency markers and are capable of robust hemangioblastdifferentiation in accordance with an embodiment of the presentinvention. After 4-5 passages under feeder-free conditions WA01 cellswere stained for expression of the hESC markers Oct-4 (A-C: DAPI, Oct-4and merged respectively) and Tra-1-60 (D-F DAPI, TRA-1-60, and mergedrespectively) Panels G and H demonstrate differences in colonymorphology when hESCs are cultured on Matrigel (G) verses MEFs (H).Magnification: originally X100. In panel I, hESCs were grown either onMEFs or Matrigel and then differentiated under the optimized conditionsdescribed herein. Considerably more hemangioblast expansion was observedin Matrigel cultured cells as compared to MEF cultured hESCs. *P<0.03,n=3.

FIG. 13 depicts qRT-PCR analysis of gene expression in EBs culturedunder different conditions in accordance with an embodiment of thepresent invention. Expression levels of various genes associated withdevelopment of hemangioblasts were analyzed in EBs derived in thepresence or absence of either or a combination of both BMP-4 andVEGF₁₆₅. β-Actin was used as an internal control to normalize geneexpression. Relative gene expression is presented as a fold differencecompared to average expression levels observed in undifferentiatedhESCs. **P<0.002; ***P<0.0004, n=3.

FIG. 14 depicts identification of surface markers for hemangioblastprogenitors in accordance with an embodiment of the present invention.EB cells were enriched with different antibodies using EasySep Kit, thenplated for the development of blast colonies. **P<0.01, n=3.

FIG. 15 depicts a wild-type nucleic acid sequence of HOXB4 protein inaccordance with an embodiment of the present invention.

FIG. 16 depicts a wild-type nucleic acid sequence of HOXB4 protein inaccordance with an embodiment of the present invention.

FIG. 17 depicts an amino acid sequence of HOXB4 in accordance with anembodiment of the present invention.

FIG. 18 depicts an amino acid sequence of HOXB4 in accordance with anembodiment of the present invention.

FIG. 19 depicts iPSCs (IMR90-1) grown under feeder-free conditionsretain pluripotency markers in accordance with an embodiment of thepresent invention. After 4-5 passages under feeder-free conditionsiPS(IMR90)-1 cells were stained for expression of pluripotency markers.a, bright field; b, Nanog; c, Oct-4; d, SSEA-4; and e, TRA-1-60.Magnification: originally X200.

FIG. 20 depicts the effect of ROCK inhibitor on IPSO hemangioblasticdifferentiation in accordance with an embodiment of the presentinvention. EBs generated from iPS(IMR90)-1 cells 24 hr after platingwithout (a, originally 100×) and with (b, originally 100×) ROCKinhibitor; Blast colonies derived from iPS(IMR90)-1 cells without ROCKinhibitor (c, originally 200×), with ROCK inhibitor (d, originally200×), and with ROCK inhibitor plus Art pathway inhibitor (e, originally200×) during EB formation; ; f-j: Hematopoietic and endothelial celldifferentiation of iPSC-derived hemangioblasts: f (originally200×);CFU-E; g (originally 100×), CFU-M; h (originally 40×), CFU-G; i(originally 400×), uptake of Ac-LDL (red) by endothelial cells stainedwith VE-Cadherin (green); j (originally 40×), tube-like network afterplating endothelial cells on Matrigel.

FIG. 21 depicts characterization of hESC-derived megakaryocytes inaccordance with an embodiment of the present invention. A. FACS analysisof cells from day 4 megakaryocyte maturation cultures. Cells werestained with megakaryocyte markers CD41a, CD42b and erythroid lineagemarker CD235a. B. FACS analysis of DNA content (Propidium iodidestaining) of gated CD41a+ megakaryocytes from day 6 maturation culture.The intensity of PI staining is shown in log scale. C. A May-grunwaldgiemsa stained mature polyploid megakaryocyte. D. Immuno-fluorescentstaining of a mature polyploid megakaryocyte with CD41 (green) and VWF(red) from the cytospin preparation of day 6 megakaryocyte maturationculture. E. A phase contrast image shows proplatelet formingmegakaryocytes (red arrows) in day 7 liquid maturation culture.

FIG. 22 depicts FACS analysis of in vitro hESC derived platelets inaccordance with an embodiment of the present invention. Human peripheralblood platelets were used as controls. CD41a+ particles derived fromhESCs are of similar FSC and SSC characteristics of peripheral bloodplatelets.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3^(rd) ed., J. Wiley & Sons (New York, N.Y. 2001); March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^(th)ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russel,Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled inthe art with a general guide to many of the terms used in the presentapplication.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers.

The term “embryonic stem cells” (ES cells) refers to embryo-derivedcells and is used herein as it is used in the art. This term includescells derived from the inner cell mass of human blastocysts or morulae,including those that have been serially passaged as cell lines. Whenused to refer to cells from humans, the term human embryonic stem cell(hES) cell is used. The ES cells may be derived from fertilization of anegg cell with sperm, as well as using DNA, nuclear transfer,parthenogenesis, or by means to generate ES cells with homozygosity inthe HLA region. ES cells are also cells derived from a zygote,blastomeres, or blastocyst-staged mammalian embryo produced by thefusion of a sperm and egg cell, nuclear transfer, parthenogenesis,androgenesis, or the reprogramming of chromatin and subsequentincorporation of the reprogrammed chromatin into a plasma membrane toproduce a cell. Embryonic stem cells, regardless of their source or theparticular method use to produce them, can be identified based on (i)the ability to differentiate into cells of all three germ layers, (ii)expression of at least Oct-4 and alkaline phosphatase, and (iii) abilityto produce teratomas when transplanted into immunodeficient animals.

As used herein, the term “pluripotent stem cells” includes embryonicstem cells, embryo-derived stem cells, and induced pluripotent stemcells, regardless of the method by which the pluripotent stem cells arederived. Pluripotent stem cells are defined functionally as stem cellsthat are: (a) capable of inducing teratomas when transplanted inimmunodeficient (SCID) mice; (b) capable of differentiating to celltypes of all three germ layers (e.g., can differentiate to ectodermal,mesodermal, and endodermal cell types); and (c) express one or moremarkers of embryonic stem cells (e.g., express Oct 4, alkalinephosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog,TRA-1-60, TRA-1-81, SOX2, REX1, etc). Exemplary pluripotent stem cellscan be generated using, for example, methods known in the art. Exemplarypluripotent stem cells include embryonic stem cells derived from the ICMof blastocyst stage embryos, as well as embryonic stem cells derivedfrom one or more blastomeres of a cleavage stage or morula stage embryo(optionally without destroying the remainder of the embryo). Suchembryonic stem cells can be generated from embryonic material producedby fertilization or by asexual means, including somatic cell nucleartransfer (SCNT), parthenogenesis, and androgenesis. Further exemplarypluripotent stem cells include induced pluripotent stem cells (iPScells) generated by reprogramming a somatic cell by expressing acombination of factors (herein referred to as reprogramming factors).iPS cells can be generated using fetal, postnatal, newborn, juvenile, oradult somatic cells. In certain embodiments, factors that can be used toreprogram somatic cells to pluripotent stem cells include, for example,a combination of Oct4 (sometimes referred to as Oct 3/4), Sox2, c-Myc,and Klf4. In other embodiments, factors that can be used to reprogramsomatic cells to pluripotent stem cells include, for example, acombination of Oct 4, Sox2, Nanog, and Lin28. In other embodiments,somatic cells are reprogrammed by expressing at least 2 reprogrammingfactors, at least three reprogramming factors, or four reprogrammingfactors. In other embodiments, additional reprogramming factors areidentified and used alone or in combination with one or more knownreprogramming factors to reprogram a somatic cell to a pluripotent stemcell. Induced pluripotent stem cells are defined functionally andinclude cells that are reprogrammed using any of a variety of methods(integrative vectors, non-integrative vectors, chemical means, etc).

The pluripotent stem cells can be from any species. Embryonic stem cellshave been successfully derived in, for example, mice, multiple speciesof non-human primates, and humans, and embryonic stem-like cells havebeen generated from numerous additional species. Thus, one of skill inthe art can generate embryonic stem cells and embryo-derived stem cellsfrom any species, including but not limited to, human, non-humanprimates, rodents (mice, rats), ungulates (cows, sheep, etc), dogs(domestic and wild dogs), cats (domestic and wild cats such as lions,tigers, cheetahs), rabbits, hamsters, gerbils, squirrel, guinea pig,goats, elephants, panda (including giant panda), pigs, raccoon, horse,zebra, marine mammals (dolphin, whales, etc.) and the like. In certainembodiments, the species is an endangered species. In certainembodiments, the species is a currently extinct species.

Similarly, iPS cells can be from any species. iPS cells have beensuccessfully generated using mouse and human cells. iPS cells have beensuccessfully generated using embryonic, fetal, newborn, and adulttissue. Accordingly, one can readily generate iPS cells using a donorcell from any species. Thus, one can generate iPS cells from anyspecies, including but not limited to, human, non-human primates,rodents (mice, rats), ungulates (cows, sheep, etc), dogs (domestic andwild dogs), cats (domestic and wild cats such as lions, tigers,cheetahs), rabbits, hamsters, goats, elephants, panda (including giantpanda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales,etc.) and the like. In certain embodiments, the species is an endangeredspecies. In certain embodiments, the species is a currently extinctspecies.

Induced pluripotent stem cells can be generated using, as a startingpoint, virtually any somatic cell of any developmental stage. Forexample, the cell can be from an embryo, fetus, neonate, juvenile, oradult donor. Exemplary somatic cells that can be used includefibroblasts, such as dermal fibroblasts obtained by a skin sample orbiopsy, synoviocytes from synovial tissue, foreskin cells, cheek cells,or lung fibroblasts. Although skin and cheek provide a readily availableand easily attainable source of appropriate cells, virtually any cellcan be used. In certain embodiments, the somatic cell is not afibroblast.

Note that the pluripotent stem cells can be, for example, ES cells orinduced pluripotent stem cells. Induced pluripotent stem cells can beproduced by expressing a combination of reprogramming factors in asomatic cell. In certain embodiments, at least two reprogramming factorsare expressed in a somatic cell to successfully reprogram the somaticcell. In other embodiments, at least three reprogramming factors areexpressed in a somatic cell to successfully reprogram the somatic cell.In other embodiments, at least four reprogramming factors are expressedin a somatic cell to successfully reprogram the somatic cell.

The term “protein transduction domain” (“PTD”) refers to any amino acidsequence that translocates across a cell membrane into cells or confersor increases the rate of, for example, another molecule (such as, forexample, a protein domain) to which the PTD is attached, to translocateacross a cell membrane into cells. The protein transduction domain maybe a domain or sequence that occurs naturally as part of a largerprotein (e.g., a PTD of a viral protein such as HIV TAT) or may be asynthetic or artificial amino acid sequence.

The terms “hemangioblast” and “hemangio-colony forming cell” will beused interchangeably throughout this application. The cells havenumerous structural and functional characteristics. Amongst thecharacteristics of these cells is the ability to engraft into the bonemarrow when administered to a host. These cells can be described basedon numerous structural and functional properties including, but notlimited to, expression (RNA or protein) or lack of expression (RNA orprotein) of one or more markers. Hemangio-colony forming cells arecapable of differentiating to give rise to at least hematopoietic celltypes or endothelial cell types. Hemangio-colony forming cells arepreferably bi-potential and capable of differentiating to give rise toat least hematopoietic cell types and endothelial cell types. As such,hemangio-colony forming cells of the present invention are at leastuni-potential, and preferably bi-potential. Additionally however,hemangio-colony forming cells may have a greater degree of developmentalpotential and can, in certain embodiments, differentiate to give rise tocell types of other lineages. In certain embodiments, thehemangio-colony forming cells are capable of differentiating to giverise to other mesodermal derivatives such as cardiac cells (for example,cardiomyocytes) and/or smooth muscle cells.

The term “non-engrafting hemangio cells” is used throughout thisapplication to refer to a novel population of cells that share some ofthe characteristics of hemangio-colony forming cells. However, thenon-engrafting hemangio cells are distinguishable in that they do notengraft into the bone marrow when administered to an immunodeficienthost. Despite this difference, non-engrafting hemangio cells may shareone or more than one (2, 3, 4, 5, 6, 7, 8, 9, 10) of the functional orstructural characteristics/properties of hemangio-colony forming cells.For example, in certain embodiments, the non-engrafting hemangio cellsare loosely adherent to each other. In other embodiments, thenon-engrafting hemangio cells do not express one or more than one (2, 3,4) of the following proteins: CD34, KDR, CD133, CD31. Without beingbound by theory, non-engrafting hemangio cells may provide a distinctstem cell population that is somewhat more committed thanhemangio-colony forming cells, and yet still capable of producing arange of hematopoietic cell types.

Enucleated Erythroid Cells

Embodiments of present invention generally relates to methods fordifferentiating human pluripotent stem cells into enucleated erythroidcells. Erythroid cells of the invention have a variety of uses in vitroand in vivo. Red blood cells of the invention will be useful in varioustherapeutic applications. Furthermore, the expanded numbers of red bloodcells derived by the present invention may be utilized in noveltherapeutic strategies in the treatment of hematopoietic disorders or inblood banking.

In certain embodiments of the application pluripotent stem cells arehemangioblasts (e.g., hemangioblasts, hemangio colony forming cells,non-engrafting hemangio cells, hemangio cells, or blast cells, see e.g.,US Patent Application 2008/0014180, herein incorporated by reference inits entirety).

In certain embodiments, the red blood cells of the application may beused in transfusions. The ability to generate large numbers of cells fortransfusion will alleviate the chronic shortage of blood experienced inblood banks and hospitals across the country. In certain embodiments,the methods of the invention allow for the production of universal cellsfor transfusion. Specifically, red blood cells that are type O and Rh—can be readily generated and will serve as a universal blood source fortransfusion.

The methods of this invention allow for the in vitro expansion ofpluripotent stem cells to large quantities useful for a variety ofcommercial and clinical applications. In certain embodiments, the cellpreparations comprise at least 1×10⁶ cells. In other embodiments, thecell preparations comprise at least 2×10⁶ human pluripotent stem cellsand in further embodiments at least 3×10⁶ human pluripotent stem cells.In still other embodiments, the cell preparations comprise at least4×10⁶ human pluripotent stem cells.

The present invention relates to a solution, a preparation, and acomposition comprising between 10,000 and 4 million or more mammalian(such as human) hemangioblast cells. The number of hemangioblast cellsin such a solution, a preparation, and a composition may be any numberbetween the range of 10,000 to 4 million, or more. This number could be,for example, 20,000, 50,000, 100,000, 500,000, 1 million, etc.

Similarly, the invention relates to preparations of red blood cells. Theinvention further relates to methods of producing, storing, anddistributing pluripotent stem cells and/or red blood cells.

The invention also provides methods and solutions suitable fortransfusion into human or animal patients. In particular embodiments,the invention provides methods of making red blood cells. In certainembodiments, the invention is suitable for use in blood banks andhospitals to provide blood for transfusion following trauma, or in thetreatment of a blood-related disease or disorder. In certainembodiments, the invention provides red blood cells that are universaldonor cells. In certain embodiments, the red blood cells are functionaland express hemoglobin F prior to transfusion.

In certain embodiments, red blood cells are transfused to treat trauma,blood loss during surgery, or blood diseases such as anemia, Sickle cellanemia, or hemolytic disease. In certain embodiments, differentiated redblood cells are transfused to treat trauma, blood loss during surgery,blood diseases such as anemia, Sickle cell anemia, or hemolyticdiseases, or malignant disease. In certain embodiments, a mixedpopulation of red blood cells is transfused. It should be noted thatmany differentiated hematopoietic cell types, particularly red bloodcells, typically exist in vivo as a mixed population. Specifically,circulating red blood cells of varying levels of age and differentiationare found in vivo. Additionally, red blood cells mature over time so asto express less fetal hemoglobin and more adult hemoglobin. The presentinvention contemplates transfusion of either purified populations of redblood cells or of a mixed population of red blood cells having varyinglevels of age and levels of differentiation. In particular embodiments,the invention contemplates transfusion of red blood cells expressingfetal hemoglobin (hemoglobin F). Transfusion of red blood cells thatexpress fetal hemoglobin may be especially useful in the treatment ofSickle cell anemia. The ability to generate large numbers of cells fortransfusion will alleviate the chronic shortage of blood experienced inblood banks and hospitals across the country.

In certain embodiments, the methods of the invention allow for theproduction of universal cells for transfusion. Specifically, red bloodcells that are type O and Rh— can be readily generated and will serve asa universal blood source for transfusion. In certain embodiments, thered blood cells produced from the methods of the application arefunctional. in certain embodiments, the red blood cells expresshemoglobin F prior to transfusion. In certain embodiments, the red bloodcells carry oxygen. In certain embodiments, the red blood cells have alifespan equal to naturally derived red blood cells. In certainembodiments, the red blood cells have a lifespan that is 75% of that ofnaturally derived red blood cells. In certain embodiments, the red bloodcells have a lifespan that is 50% of that of naturally derived red bloodcells. In certain embodiments, the red blood cells have a lifespan thatis 25% of that of naturally derived red blood cells.

The differentiation of stem cells into mature red blood cells is acurrent challenge. The impact of this achievement is enormous, as thereis a constant blood donor shortage, with inconsistent supply and highdemand, especially in times of unexpected crisis situations. Embryonicstem cells (ESCs) are a potential consistent and reliable source of redblood cells, with the benefits of unlimited supply of O-universal blood,and avoiding the additional cost of disease screening and blood typingwith each donation. The hallmark of mature red blood cells is loss ofthe nucleus, as well as production of mature hemoglobin. Manyresearchers, including our laboratory, have achieved differentiation ofESCs into erythroblasts, which still contain their nuclei, and expressimmature hemoglobin. To date, enucleation has not been achieved withhuman embryonic stem cells.

By contrast, enucleation has been achieved with CD34+ bone marrow andcord blood stem cells, which are further along in development, thusprobably aiding in their enucleation capability. Malik achieved 10-40%enucleation after 19 days of treatment of CD34+ bone marrow cells withEpo (Malik 1998). Miharada achieved a rate of 77% enucleation from CD34+cord blood stem cells with a 20-day treatment of growth factors andcytokines including SCF, Epo, IL-3, VEGF, IGF-II, and mifepristone(Miharada 2006). Douay achieved an even higher enucleation rate in CD34+cord blood stem cells of 90-100% with an 18-day protocol in a cocktailof factors found in the bone marrow environment (SCF, IL-3, Epo,hydrocortisone), with the addition of co-culturing the cells with MS-5mouse stromal line or mesenchymal stem cells (MSCs) (Douay 2005).Although growth factors are used to mimic the environment of the bonemarrow niche in which erythroblasts mature, cell contacts may also benecessary to signal enucleation, as shown by the abrogation ofenucleation when cord blood and stromal cells were separated fromphysical contact but grown in the same media. Although successful forcord blood stem cells, these protocols fail to produce enucleation inESCs.

In certain embodiments, the present inventive method uses OP9 cells toinduce differentiation in human ESCs in a completely in vitro system,which is relevant to clinical therapies. In certain embodiments, thefirst step consists of differentiating ESCs into hemangioblasts,hemangio colony forming cells, non-engrafting hemangio cells, or blastcells. In certain embodiments, the second step is expansion of thesecells in Stemline II medium (Sigma) with Epo, IL-3, SCF and varioussupplements used by Douay for cord blood enucleation (Douay 2005). Incertain embodiments, the third step introduces the OP9 cells to theESC-derived erythroblasts, as well as the addition of Epo.

In certain embodiments, differentiating ESCs into the hemangioblasts,hemangio colony forming cells, and non-engrafting hemangio cells areproduced and expanded in accordance to methods described herein.

In certain embodiments, blast cells are cultured as described in Lu2006. In certain embodiments, day 6-8 blast cells from Day 3.25-Day 4.25embryoid bodies are picked or filtered and plated in Stemline II mediumwith Epo, IL-3, SCF, hydrocortisone, inositol, folic acid,mono-thioglycerol, transferrin, insulin, ferrous nitrate, ferroussulfate and bovine serum albumin for 12-30 days. In certain embodiments,blast cells are then co-cultured with OP9 mouse stromal cells or humanmesenchymal stem cells (MSCs) in the same media listed above, withouthydrocortisone. In certain embodiments, cells begin co-culturing betweenday 12 and 29 days. In certain embodiments, cells are further culturedfor 12-18 days before enucleation occurs. In certain embodiments,enucleation initiated by OP9 cells can occur in as little as 3 daysafter stromal growth. In certain embodiments, enucleation is induced inStempro34 medium with hydrocortisone, inositol, folic acid,mono-thioglycerol, transferrin, insulin, ferrous nitrate, ferroussulfate and bovine serum albumin. In certain embodiments, cells are fedevery 3-4 days and cultured on a new stromal layer every week.

The invention contemplates all suitable combinations of any of theforgoing or following aspects and embodiments of the invention.

Megakaryocytes and Platelets

The present invention also provides methods of producing a megakaryocyteor a platelet, comprising: providing a pluripotent stem cell;differentiating said pluripotent stem cell into a hemangioblast,non-engrafting hemangio cell, or blast cell; and differentiating saidhemangioblast, non-engrafting hemangio cell, or blast cell into saidmegakaryocyte or said platelet by culturing in megakaryocyte (MK)culture medium comprising TPO.

The present invention also provides methods of producing a megakaryocyteor a platelet, comprising: providing a hemangioblast, non-engraftinghemangio cell, or blast cell; and differentiating said hemangioblast,non-engrafting hemangio cell, or blast cell into said megakaryocyte orsaid platelet by culturing in megakaryocyte (MK) culture mediumcomprising TPO.

The hemangioblast, non-engrafting hemangio cell, or blast cell may beobtained or produced by methods described herein.

In certain embodiments, said pluripotent stem cell used in the presentinvention is an embryonic stem cell or embryo-derived cell. In certainembodiments, said pluripotent stem cell is an induced pluripotent stemcell. In certain embodiments, said pluripotent stem cell is a humancell. In certain embodiments, said pluripotent stem cell is geneticallymanipulated prior to differentiation.

In certain embodiments, said hemangioblast, non-engrafting hemangiocell, or blast cell is expanded prior to being differentiated into saidmegakaryocyte or said platelet. In certain embodiments, saidhemangioblasts, non-engrafting hemangio cells, or blast cells areexpanded in Stemline II medium with Epo, IL-3, and SCF.

In certain embodiments, differentiating said hemangioblast,non-engrafting hemangio cell, or blast cell into said megakaryocyte orsaid platelet is done after about 6 to 8 days of hemangioblast,non-engrafting hemangio cell, or blast cell culture.

The present invention also provides a megakaryocyte or a plateletproduced by any one of the method as described herein.

The methods of producing a megakaryocyte or a platelet are described inmore detail in the ensuing examples.

Hemangio-Colony Forming Cells

This invention provides a method for generating and expanding humanhemangio-colony forming cells from human pluripotent stem cells,preparations and compositions comprising human hemangio-colony formingcells, methods of producing various cell types partially or terminallydifferentiated from hemangio-colony forming cells, methods of usinghemangio-colony forming cells therapeutically, and methods oftherapeutically using various cell types partially or terminallydifferentiated from hemangio-colony forming cells.

Here, the inventors report a simpler and more efficient method forrobust generation of hemangioblastic progenitors. In addition toeliminating several expensive factors that are unnecessary, it isdemonstrated that bone morphogenetic protein-4 (BMP-4) and vascularendothelial growth factor (VEGF) are necessary and sufficient to inducehemangioblastic commitment and development from pluripotent stem cellsduring early stages of differentiation. BMP-4 and VEGF significantlyup-regulate T-brachyury, KDR, CD31 and LMO2 gene expression, whiledramatically down-regulating Oct-4 expression. The addition of basicfibroblast growth factor (bFGF) during growth and expansion was found tofurther enhance BC development, consistently generating approximately1×10⁸ BCs from one six-well plate of hESCs.

This invention also provides a method for expanding mammalianhemangio-colony forming cells obtained from any source, including EScells, blastocysts or blastomeres, cord blood from placenta or umbilicaltissue, peripheral blood, bone marrow, or other tissue or by any othermeans known in the art. Human hemangio-colony forming cells can also begenerated from human pluripotent stem cells. Human pluripotent stemcells may be a substantially homogeneous population of cells, aheterogeneous population of cells, or all or a portion of an embryonictissue. As an example of pluripotent stem cells that can be used in themethods of the present invention, human hemangio-colony forming cellscan be generated from human embryonic stem cells. Such embryonic stemcells include embryonic stem cells derived from or using, for example,blastocysts, plated ICMs, one or more blastomeres, or other portions ofa pre-implantation-stage embryo or embryo-like structure, regardless ofwhether produced by fertilization, somatic cell nuclear transfer (SCNT),parthenogenesis, androgenesis, or other sexual or asexual means.

In certain embodiments, hemangioblasts can be further differentiated tohematopoietic cells including, but not limited to, platelets and redblood cells. Such cells may be used in transfusions. The ability togenerate large numbers of cells for transfusion will alleviate thechronic shortage of blood experienced in blood banks and hospitalsacross the country. In certain embodiments, the methods of the inventionallow for the production of universal cells for transfusion.Specifically, red blood cells that are type O and Rh— can be readilygenerated and will serve as a universal blood source for transfusion.

The methods of this invention allow for the in vitro expansion ofhemangioblasts to large quantities useful for a variety of commercialand clinical applications. Expansion of hemangioblasts in vitro refersto the proliferation of hemangioblasts. While the methods of theinvention enable the expansion of human hemangioblast cells to reachcommercially useful quantities, the present invention also relates tolarge numbers of hemangioblast cells and to cell preparations comprisinglarge numbers of human hemangioblast cells (for example, at least10,000, 100,000, or 500,000 cells). In certain embodiments, the cellpreparations comprise at least 1×10⁶ cells. In other embodiments, thecell preparations comprise at least 2×10⁶ human hemangioblast cells andin further embodiments at least 3×10⁶ human hemangioblast cells. Instill other embodiments, the cell preparations comprise at least 4×10⁶human hemangioblast cells.

The present invention relates to a solution, a preparation, and acomposition comprising between 10,000 and 4 million or more mammalian(such as human) hemangioblast cells. The number of hemangioblast cellsin such a solution, a preparation, and a composition may be any numberbetween the range of 10,000 to 4 million, or more. This number could be,for example, 20,000, 50,000, 100,000, 500,000, 1 million, etc.

Similarly, the invention relates to preparations of human hemangioblastprogeny cells (e.g., human hematopoietic cells including humanhematopoietic stem cells, and endothelial cells). The invention furtherrelates to methods of producing, storing, and distributing hemangioblastcells and/or hemangioblast lineage cells.

The invention also provides methods and solutions suitable fortransfusion into human or animal patients. In particular embodiments,the invention provides methods of making red blood cells and/orplatelets, and/or other hematopoietic cell types for transfusion. Incertain embodiments, the invention is suitable for use in blood banksand hospitals to provide blood for transfusion following trauma, or inthe treatment of a blood-related disease or disorder. In certainembodiments, the invention provides red blood cells that are universaldonor cells. In certain embodiments, the red blood cells are functionaland express hemoglobin F prior to transfusion.

The invention also provides for human hemangio-colony forming cells,cell cultures comprising a substantially purified population of humanhemangio-colony forming cells, pharmaceutical preparations comprisinghuman hemangio-colony forming cells and cryopreserved preparations ofthe hemangio-colony forming cells. In certain embodiments, the inventionprovides for the use of the human hemangio-colony forming cells in themanufacture of a medicament to treat a condition in a patient in needthereof. Alternatively, the invention provides the use of the cellcultures in the manufacture of a medicament to treat a condition in apatient in need thereof. The invention also provides the use of thepharmaceutical preparations in the manufacture of a medicament to treata condition in a patient in need thereof.

The hemangio-colony forming cells can be identified and characterizedbased on their structural properties. Specifically, and in certainembodiments, these cells are unique in that they are only looselyadherent to each other (loosely adherent to other hemangio-colonyforming cells). Because these cells are only loosely adherent to eachother, cultures or colonies of hemangio-colony forming cells can bedissociated to single cells using only mechanical dissociationtechniques and without the need for enzymatic dissociation techniques.The cells are sufficiently loosely adherent to each other thatmechanical dissociation alone, rather than enzymatic dissociation or acombination of mechanical and enzymatic dissociation, is sufficient todisaggregate the cultures or colonies without substantially impairingthe viability of the cells. In other words, mechanical dissociation doesnot require so much force as to cause substantial cell injury or deathwhen compared to that observed subsequent to enzymatic dissociation ofcell aggregates.

Furthermore, hemangio-colony forming cells can be identified orcharacterized based on the expression or lack of expression (as assessedat the level of the gene or the level of the protein) of one or moremarkers. For example, in certain embodiments, hemangio-colony formingcells can be identified or characterized based on lack of expression ofone or more (e.g., the cells can be characterized based on lack ofexpression of at least one, at least two, at least three or at leastfour of the following markers) of the following cell surface markers:CD34, KDR, CD133, or CD31. Additionally or alternatively,hemangio-colony forming cells can be identified or characterized basedon expression of GATA2 and/or LMO2. Additionally or alternatively,hemangio-colony forming cells can be identified or characterized basedon expression or lack of expression markers.

Hemangio-colony forming cells of the present invention can be identifiedor characterized based on one or any combination of these structural orfunctional characteristics. Note that although these cells can bederived from any of a number of sources, for example, embryonic tissue,prenatal tissue, or perinatal tissue, the term “hemangio-colony formingcells” applies to cells, regardless of source, that are capable ofdifferentiating to give rise to at least hematopoietic cell types and/orendothelial cell types and that have one or more of the foregoingstructural or functional properties.

In certain embodiments, marker(s) for the progenitor of BCs can be usedto select BCs after initial culturing.

In certain embodiments, hemangio-colonies are produced from pluripotentcells without forming embryoid bodies.

In Vitro Differentiation of Pluripotent Stem Cells to Obtain EmbryoidBodies and Hemangioblasts

The present invention provides a method for generating and expandinghuman hemangioblasts derived from human pluripotent stem cells, or fromhuman blastocysts or blastomeres. The hemangioblasts so produced may bepurified and/or isolated.

Human hemangio-colony forming cells can also be generated from humanpluripotent stem cells. Human pluripotent stem cells may be asubstantially homogeneous population of cells, a heterogeneouspopulation of cells, or all or a portion of an embryonic tissue. As anexample of pluripotent stem cells that can be used in the methods of thepresent invention, human hemangio-colony forming cells can be generatedfrom human embryonic stem cells. Such embryonic stem cells includeembryonic stem cells derived from or using, for example, blastocysts,plated ICMs, one or more blastomeres, or other portions of apre-implantation-stage embryo or embryo-like structure, regardless ofwhether produced by fertilization, somatic cell nuclear transfer (SCNT),parthenogenesis, androgenesis, or other sexual or asexual means.

Additionally or alternatively, hemangio-colony forming cells can begenerated from other pluripotent stem cells. For example,hemangio-colony forming cells can be generated (without necessarilygoing through a step of embryonic stem cell derivation) from or usingplated embryos, ICMs, blastocysts, trophoblast/trophectoderm cells, oneor more blastomeres, trophoblast stem cells, embryonic germ cells, orother portions of a pre-implantation-stage embryo or embryo-likestructure, regardless of whether produced by fertilization, somatic cellnuclear transfer (SCNT), parthenogenesis, androgenesis, or other sexualor asexual means. Similarly, hemangio-colony forming cells can begenerated using cells or cell lines partially differentiated frompluripotent stem cells. For example, if a human embryonic stem cell lineis used to produce cells that are more developmentally primitive thanhemangio-colony forming cells, in terms of development potential andplasticity, such pluripotent stem cells could then be used to generatehemangio-colony forming cells.

Additionally or alternatively, hemangio-colony forming cells can begenerated from other pre-natal or peri-natal sources including, withoutlimitation, umbilical cord, umbilical cord blood, amniotic fluid,amniotic stem cells, and placenta.

It is noted that when hemangio-colony forming cells are generated fromhuman embryonic tissue a step of embryoid body formation may be needed.However, given that embryoid body formation serves, at least in part, tohelp recapitulate the three dimensional interaction of the germ layersthat occurs during early development, such a step is not necessarilyrequired when the pluripotent stem cells already have a structure ororganization that serves substantially the same purpose as embryoid bodyformation. By way of example, when hemangio-colony forming cells aregenerated from plated blastocysts, a level of three dimensionalorganization already exists amongst the cells in the blastocyst. Assuch, a step of embryoid body formation is not necessarily required toprovide intercellular signals, inductive cues, or three dimensionalarchitecture.

The methods and uses of the present invention can be used to generatehemangio-colony forming cells from pluripotent stem cells orembryo-derived cells. In certain embodiments, the embryo-derived cellsare embryonic stem cells. In certain other embodiments, theembryo-derived cells are plated embryos, ICMs, blastocysts,trophoblast/trophectoderm cells, one or more blastomeres, trophoblaststem cells, or other portions of an early pre-implantation embryo. Forany of the foregoing, the embryo-derived cells may be from embryosproduced by fertilization, somatic cell nuclear transfer (SCNT),parthenogenesis, androgenesis, or other sexual or asexual means.

Throughout this application, when a method is described by referringspecifically to generating hemangio-colony forming cells from embryonicstem cells, the invention similarly contemplates generatinghemangio-colony forming cells from or using other pluripotent stem cellsor embryonic-derived cells, and using the generated cells for any of thesame therapeutic applications.

In certain aspects of the invention, the human embryonic stem cells maybe the starting material of this method. The embryonic stem cells may becultured in any way known in the art, such as in the presence or absenceof feeder cells.

Embryonic stem cells may form embryoid bodies (“EBs”) in suspension inmedium containing serum (Wang et al. 2005 J Exp Med (201):1603-1614;Wang at al. 2004 Immunity (21): 31-41; Chadwick et al. 2003 Blood (102):906-915). The addition of serum, however, presents certain challenges,including variability in experiments, cost, potential for infectiousagents, and limited supply. Further, for clinical and certain commercialapplications, use of serum necessitates additional U.S. andinternational regulatory compliance issues that govern biologicalproducts.

The present invention provides methods of generating and expanding humanhemangioblasts from pluripotent stem cells in which no serum is used.The serum-free conditions are more conducive to scale-up productionunder good manufacturing process (GMP) guidelines than are conditionswhich require serum. Furthermore, serum-free conditions extend thehalf-life of certain factors added to the medium (for example, thehalf-life of proteins including growth factors, cytokines, and HOXB4 inmedia is increased when no serum is present). In certain embodiments,the media is supplemented with BMP4 and VEGF. In certain embodiments,serum-free media is used throughout the method of this invention forgenerating and expanding human hemangioblasts.

In the first step of this method for generating and expanding humanhemangioblast cells, human stem cells are grown in serum-free media andare induced to differentiate into embryoid bodies. To induce embryoidbody formation, embryonic stem cells may be pelleted and resuspended inserum-free medium (e.g., in Stemline I or II media (Sigma™))supplemented with one or more morphogenic factors and cytokines and thenplated on low attachment (e.g., ultra-low attachment) culture dishes.Morphogenic factors and cytokines may include, but are not limited to,bone morphogenic proteins (e.g., BMP2, BMP-4, BMP-7, but not BMP-3) andVEGF, SCF and FL. Bone morphogenic proteins and VEGF may be used aloneor in combination with other factors. The morphogenic factors andcytokines may be added to the media from 0-48 hours of cell culture.Following incubation under these conditions, incubation in the presenceof early hematopoietic expansion cytokines, including, but not limitedto, thrombopoietin (TPO), Flt-3 ligand, and stem cell factor (SCF),allows the plated ES cells to form EBs. In addition to TPO, Flt-3ligand, and SCF, VEGF, BMP-4, and HoxB4 may also be added to the media.In one embodiment, human ES cells are first grown in the presence ofBMP-4 and VEGF₁₆₅ (e.g., 25-100 ng/ml), followed by growing in thepresence of BMP-4, VEGF₁₆₅, SCF, TPO, and FLT3 ligand (e.g., 10-50ng/ml) and HoxB4 (e.g., 1.5-5 μg/ml of a triple protein transductiondomain-HoxB4 fusion protein as disclosed herein). The additional factorsmay be added 48-72 hours after plating.

In this method of the present invention, human hemangioblast cells areisolated from early embryoid bodies (“EBs”). Isolating hemangioblastcells from early EBs supports the expansion of the cells in vitro. Forhuman cells, hemangioblast cells may be obtained from EBs grown for lessthan 10 days. In certain embodiments of the present invention,hemangioblast cells arise in human EBs grown for 2-6 days. According toone embodiment, hemangioblast cells are identified and may be isolatedfrom human EBs grown for 4-6 days. In other embodiments, human EBs aregrown for 2-5 days before hemangioblast cells are isolated. In certainembodiments, human EBs are grown for 3-4.5 days before hemangioblastcells are isolated.

In certain embodiments, early EBs are washed and dissociated (e.g., byTrypsin/EDTA or collagenase B). A select number of cells (e.g., 2-5×10⁵cells) are then mixed with serum-free methylcellulose medium optimizedfor hemangioblast cell growth (e.g., BL-CFU medium, for example StemCell Technologies Catalogue H4436, or hemangioblast cell expansionmedium (HGM), or any medium containing 1.0% methylcellulose in MDM, 1-2%Bovine serum albumin, 0.1 mM 2-mercaptoethanol, 10 μg/ml rh-Insulin, 200μg/ml iron saturated human transferrin, 20 ng/ml rh-GM-CSF, 20 ng/mlrh-IL-3, 20 ng/ml rh-IL-6, 20 ng/ml rh-G-CSF) (“rh” stands for“recombinant human”). This medium may be supplemented with early stagecytokines (including, but not limited to, EPO, TPO, SCF, FL, FLt-3,VEGF, BMPs such as BMP2, BMP4 and BMP7, but not BMP3) and HOXB4 (oranother homeobox protein). In certain embodiments, erythropoietin (EPO)is added to the media. In further embodiments, EPO, SCF, VEGF, BMP-4 andHoxB4 are added to the media. In additional embodiments, the cells aregrown in the presence of EPO, TPO and FL. In certain embodiments whereH9 is the starting human ES cell line, EPO, TPO and FL are added to themedia. In addition to EPO, TPO and FL, media for cells derived from H9or other ES cells may further comprise VEGF, BMP-4, and HoxB4.

The cells so obtained by this method (the cells may be in BL-CFUmedium), which include hemangioblast cells, are plated onto ultra-lowattachment culture dishes and incubated in a CO₂ incubator to growhemangioblast colonies. Some cells may be able to form secondary EBs.Following approximately 3-6 days, and in some instances 3-4.5 days,hemangioblast colonies are observed. Hemangioblast colonies may bedistinguished from other cells such as secondary EBs by theirdistinctive grape-like morphology and/or by their small size. Inaddition, hemangioblasts may be identified by the expression of certainmarkers (e.g., the expression of both early hematopoietic andendothelial cell markers) as well as their ability to differentiate intoat least both hematopoietic and endothelial cells (see below, Derivinghemangioblast lineage cells). For example, while hemangioblasts lackcertain features characteristic of mature endothelial or hematopoieticcells, these cells may be identified by the presence of certain markers(such as, for example, CD71+) and the absence of other markers (forexample, CD34−). Hemangioblasts may also express GATA-1 and GATA-2proteins, CXCR-4, and TPO and EPO receptors. In addition, hemangioblastsmay be characterized by the absence or low expression of other markers(e.g., CD31, CD34, KDR, or other adhesion molecules). Further,hemangioblasts may be characterized by the expression of certain genes,(e.g., genes associated with hemangioblasts and early primitiveerythroblast development, such as, for example, SCL, LMO2, FLT-1,embryonic fetal globin genes, NF-E2, GATA-1, EKLF, ICAM-4,glycophoriuns, and EPO receptor).

Accordingly, hemangioblasts may be isolated by size (being smaller thanthe other cells) or purified with an anti-CD71+ antibody, such as byimmunoaffinity column chromatography.

The hemangioblast cells may be isolated by size and/or morphology by thefollowing procedure. After 6 to 7 days of growth, the cell mixturecontains EBs, which are round and represent a clump of multiple cells,and hemangioblasts, which are grape-like, smaller than the EBs, and aresingle cells. Accordingly, hemangioblasts may be isolated based on theirmorphology and size. The hemangioblast cells may be manually picked, forexample, when observing the cell mixture under a microscope. The cellsmay subsequently grow into colonies, each colony having between 100-150cells.

Human hemangioblast colonies derived as described above may be pickedand replated onto methylcellulose CFU-medium to form hematopoietic CFUs.In certain embodiments, CFU-medium comprises StemCell TechnologiesH4436. In further embodiments, hemangioblasts are plated in Stemline IImedia supplemented with cytokines and other factors. For example,individual BL-CFC colonies may be handpicked and transferred to afibronectin-coated plate containing Stemline II with recombinant humanSCF (e.g., 20 ng/ml), TPO (e.g., 20 ng/ml), FL (e.g., 20 ng/ml), IL-3(e.g., 20 ng/ml) VEGF (e.g., 20 ng/ml), G-CSF (e.g., 20 n ng/ml), BMP-4(e.g., 15 ng/ml), IL-6 (e.g., 10 ng/ml), IGF-1 (e.g., 10 ng/ml),endothelial cell growth supplement (ECGS, e.g., 100 pg/ml), Epo (e.g., 3U/ml). Following one week of growth in vitro, non-adherent hematopoieticcells may be removed by gentle pipetting and used directly forhematopoietic CFU assay. Following removal of the non-adherent cells,the adherent populations may be grown for one more week in EGM-2endothelial cell medium (Cambrex™), and then examined for the expressionof vWF.

Expansion of Hemangioblasts In Vitro

Certain aspects of the invention relate to the in vitro expansion ofhemangioblasts. In certain embodiments, hemangioblasts expanded by themethods of the invention are obtained from early embryoid bodies derivedfrom human embryonic stem cells as described above.

In addition to deriving hemangioblasts from human embryonic stem cells(hES cells), hemangioblasts to be expanded may also be isolated fromother mammalian sources, such as mammalian embryos (Ogawa et al. 2001Int Rev Immunol (20):21-44, US patent publication no. 2004/0052771),cord blood from placenta and umbilical tissues (Pelosi, et al. 2002Blood (100): 3203-3208; Cogle et al. 2004 Blood (103):133-5), peripheralblood and bone marrow (Pelosi et al. 2002 Hematopoiesis (100):3203-3208). In certain embodiments, non-human hemangioblasts to beexpanded may be generated from non-human (such as mouse and non-humanprimates) embryonic stem cells. In certain embodiments, hemangioblastsare obtained from umbilical cord blood (UCB) or bone marrow by methodssuch as, for example, magnetic bead positive selection or purificationtechniques (e.g. MACS column). Cells may be selected based on theirCD71+ status and may be confirmed as CD34−. Further, the isolatedhemangioblasts may be tested for their potential to give rise to bothhematopoietic and endothelial cell lineages. In certain embodiments,hemangioblasts isolated or purified and optionally enriched fromembryos, cord blood, peripheral blood, bone marrow, or other tissue, aremore than 95% pure.

Bone marrow-derived cells may be obtained from any stage of developmentof the donor individual, including prenatal (e.g., embryonic or fetal),infant (e.g., from birth to approximately three years of age in humans),child (e.g., from about three years of age to about 13 years of age inhumans), adolescent (e.g., from about 13 years of age to about 18 yearsof age in humans), young adult (e.g., from about 18 years of age toabout 35 years of age in humans), adult (from about 35 years of age toabout 55 years of age in humans) or elderly (e.g. from about 55 yearsand beyond of age in humans).

Human bone marrow may be harvested by scraping from the split sternum ofa patient undergoing surgery, for example. Bone marrow may then bepreserved in tissue clumps of 0.1 to 1 mm³ in volume and then grown on amouse embryonic feeder layer (e.g., a mitomycin C-treated or irradiatedfeeder layer). The bone marrow cells will attach to the plates and overa period of 1-2 weeks of culture, hemangioblast cells may be identifiedbased on morphological features and/or cell markers and isolated (see USpatent publication no. 2004/0052771). The cells may then be subsequentlygrown and expanded in serum-free conditions according to the methodsdisclosed herein.

In addition, bone marrow cells and cells from blood or other tissue maybe fractionated to obtain hemangioblasts cells. Methods of fractionationare well known in the art, and generally involve both positive selection(i.e., retention of cells based on a particular property) and negativeselection (i.e., elimination of cells based on a particular property).Methods for fractionation and enrichment of bone marrow-derived cellsare best characterized for human and mouse cells.

There are a variety of methods known in the art for fractionating andenriching bone marrow-derived or other cells. Positive selection methodssuch as enriching for cells expressing CD71 may be used. And negativeselection methods which remove or reduce cells expressing CD3, CD10,CD11b, CD14, CD16, CD15, CD16, CD19, CD20, CD32, CD45, CD45R/B220 orLy6G may also be used alone or in combination with positive selectiontechniques. In the case of bone marrow cells, when the donor bonemarrow-derived cells are not autologous, negative selection may beperformed on the cell preparation to reduce or eliminate differentiatedT cells.

Generally, methods used for selection/enrichment of bone marrow-derived,blood, or other cells will utilize immunoaffinity technology, althoughdensity centrifugation methods are also useful. Immunoaffinitytechnology may take a variety of forms, as is well known in the art, butgenerally utilizes an antibody or antibody derivative in combinationwith some type of segregation technology. The segregation technologygenerally results in physical segregation of cells bound by the antibodyand cells not bound by the antibody, although in some instances thesegregation technology which kills the cells bound by the antibody maybe used for negative selection.

Any suitable immunoaffinity technology may be utilized forselection/enrichment of hemangioblasts from bone marrow-derived, blood,or other cells, including fluorescence-activated cell sorting (FAGS),panning, immunomagnetic separation, immunoaffinity chromatography,antibody-mediated complement fixation, immunotoxin, density gradientsegregation, and the like. After processing in the immunoaffinityprocess, the desired cells (the cells bound by the immunoaffinityreagent in the case of positive selection, and cells not bound by theimmunoaffinity reagent in the case of negative selection) are collectedand may be subjected to further rounds of immunoaffinityselection/enrichment.

Immunoaffinity selection/enrichment is typically carried out byincubating a preparation of cells comprising bone marrow-derived cellswith an antibody or antibody-derived affinity reagent (e.g., an antibodyspecific for a given surface marker), then utilizing the bound affinityreagent to select either for or against the cells to which the antibodyis bound. The selection process generally involves a physicalseparation, such as can be accomplished by directing droplets containingsingle cells into different containers depending on the presence orabsence of bound affinity reagent (FACS), by utilizing an antibody bound(directly or indirectly) to a solid phase substrate (panning,immunoaffinity chromatography), or by utilizing a magnetic field tocollect the cells which are bound to magnetic particles via the affinityreagent (immunomagnetic separation). Alternatively, undesirable cellsmay be eliminated from the bone marrow-derived cell preparation using anaffinity reagent which directs a cytotoxic insult to the cells bound bythe affinity reagent. The cytotoxic insult may be activated by theaffinity reagent (e.g., complement fixation), or may be localized to thetarget cells by the affinity reagent (e.g., immunotoxin, such as ricin Bchain).

Although the methods described above refer to enrichment of cells from apreparation of bone marrow-derived or blood cells, one skilled in theart will recognize that similar positive and negative selectiontechniques may be applied to cell preparations from other tissues.

Certain aspects of the invention relate to the in vitro expansion ofhemangioblasts. In certain embodiments, hemangioblasts expanded by themethods of the invention are obtained from early embryoid bodies derivedfrom human embryonic stem cells as described above. In otherembodiments, the hemangioblasts are isolated or enriched from humantissue (e.g., placenta or cord blood, peripheral blood, bone marrow,etc.)

In certain embodiments, the hemangioblasts are expanded in the presenceof a homeodomain protein (also referred to herein as a homeoboxprotein). In further embodiments, the hemangioblasts are expanded in thepresence of HOXB4. In certain embodiments, HOXB4 is added to thehemangioblast cells throughout the method for expanding hemangioblastcells.

HOXB4 is a homeodomain transcription factor (also called HOX2F, HOX2,HOX-2.6, and in the rat HOXA5) that is expressed in vivo in the stemcell fraction of the bone marrow and that is subsequently down-regulatedduring differentiation. Expression of the HOXB4 gene is associated withthe maintenance of primitive stem cell phenotypes (Sauvageau et al. 1995Genes Dev 9: 1753-1765; Buske et al. 2002 Blood 100: 862-868;Thorsteinsdottir et al. 1999 Blood 94: 2605-2612; Antonchuk of al. 2001Exp Hematol 29: 1125-1134).

HOXB4 used in the methods of the present invention to generate andexpand hemangioblasts, includes, but is not limited to, full lengthHOXB4 (e.g., HOXB4 polypeptides specified by public accession numbersGI:13273315 (FIG. 17), GI:29351568 (FIG. 18), as well as any functionalvariants and active fragments thereof. The wild-type HOXB4 protein maybe encoded by the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3 orany other alternative allelic forms of such protein. Such sequences maybe accessed via publicly available databases, such as Genbank. Further,HOXB4 may be ectopically expressed within the cell or may be provided inthe media. HOXB4 expressed ectopically may be operably linked to aninducible promoter. HOXB4 provided in the media may be excreted byanother cell type (e.g., a feeder layer) or added directly to the media.

The present invention also relates to fusion proteins comprising HOXB4(including fusion proteins comprising full length HOXB4, or HOXB4functional variants or active fragments of HOXB4). In addition to HOXB4,this fusion protein may also comprise any additional proteins, proteindomains or peptides. In certain embodiments, HOXB4 may be joined to aprotein transduction domain (PTD) to allow translocation of the proteinfrom the medium into the cells and subsequently into nuclearcompartments. Fusion proteins may or may not comprise one or more linkersequences located in between the protein domains.

Functional variants of HOXB4 include mutants of HOXB4 and allelicvariants, and active fragments thereof. Functional variants of HOXB4include any HOXB4 polypeptides and active fragments thereof that arecapable of expanding hemangioblasts according to the methods of thepresent invention. HOXB4 functional variants also include HOXB4polypeptides that exhibit greater transcriptional activity compared tothe native HOXB4 protein. HOXB4 variants include proteins with one ormore amino acid substitution, addition, and/or deletion in relation to awild-type HOXB4. HOXB4 variants also include, but are not limited to,polypeptides that are at least 75% similar to the sequence provided inSEQ ID NO: 1 or SEQ ID NO: 3. Accordingly, HOXB4 variants includepolypeptides that are 80%, 85%, 90%, 95%, and 99% similar to the aminoacid sequence provided in SEQ ID NO: 1 or SEQ ID NO: 3.

HOXB4 variants also include polypeptides encoded by nucleic acidsequences that are at least 80% identical to a nucleic acid sequenceencoding its complement (e.g., the wild-type HOXB4 protein may beencoded by nucleic acid sequences of SEQ ID NO: 2 (GI:85376187; FIG. 15)or SEQ ID NO: 4 (GI:29351567; FIG. 16)). Thus, HOXB4 variants includeHOXB4 polypeptides that are encoded by nucleic acid sequences that are85%, 90%, 95%, and 99% identical to the sequence provided in SEQ ID NO:2 or SEQ ID NO: 4 or complement thereto.

Nucleic acid sequences encoding HOXB4 also include, but are not limitedto, any nucleic acid sequence that hybridizes under stringent conditionsto a nucleic acid sequence of SEQ ID NO: 2 or 4, complement thereto, orfragment thereof. Similarly, nucleic acids which differ from the nucleicacids as set forth in SEQ ID NO: 2 or 4 due to degeneracy in the geneticcode are also within the scope of the invention. HOXB4 variantpolypeptides also include splice variants or other naturally occurringHOXB4 proteins or nucleic acid sequences.

Active fragments of HOXB4 include, but are not limited to, any fragmentof full length HOXB4 polypeptide that is capable of maintaininghemangioblasts according to the methods of the present invention.Accordingly, in one embodiment, a HOXB4 protein of the present inventionis a HOXB4 protein that lacks part of the N-terminus, such as, forexample, the N-terminal 31,32, or 33 amino acids of full length HOXB4.

Any of the HOXB4 proteins may be fused with additional proteins orprotein domains. For example, HOXB4 may be joined to a proteintransduction domain (PTD).

Protein transduction domains, covalently or non-covalently linked toHOXB4, allow the translocation of HOXB4 across the cell membranes so theprotein may ultimately reach the nuclear compartments of the cells.

PTDs that may be fused with a HOXB4 protein include the PTD of the HIVtransactivating protein (TAT) (Tat 47-57) (Schwarze and Dowdy 2000Trends Pharmacol. Sci. 21: 45-48; Krosl et al. 2003 Nature Medicine (9):1428-1432). For the HIV TAT protein, the amino acid sequence conferringmembrane translocation activity corresponds to residues 47-57(YGRKKRRQRRR, SEQ ID NO: 5) (Ho et al., 2001, Cancer Research 61:473-477; Vives et al., 1997, J. Biol. Chem. 272: 16010-16017). Thissequence alone can confer protein translocation activity. The TAT PTDmay also be the nine amino acids peptide sequence RKKRRQRRR (SEQ ID NO:6) (Park et al. Mol Cells 2002 (30):202-8). The TAT PTD sequences may beany of the peptide sequences disclosed in Ho et al., 2001, CancerResearch 61: 473-477 (the disclosure of which is hereby incorporated byreference herein), including YARKARRQARR (SEQ ID NO: 7), YARAAARQARA(SEQ ID NO: 8), YARAARRAARR (SEQ ID NO: 9) and RARAARRAARA (SEQ ID NO:10).

Other proteins that contain PTDs that may be fused to HOXB4 proteins ofthe present invention include the herpes simplex virus 1 (HSV-1)DNA-binding protein

VP22 and the Drosophila Antennapedia (Antp) homeotic transcriptionfactor (Schwarze et al. 2000 Trends Cell Biol. (10): 290-295). For Antp,amino acids 43-58 (RQIKIWFQNRRMKWKK, SEQ ID NO: 11) represent theprotein transduction domain, and for HSV VP22 the PTD is represented bythe residues DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 12).Alternatively, HeptaARG (RRRRRRR, SEQ ID NO: 13) or artificial peptidesthat confer transduction activity may be used as a PTD of the presentinvention.

In additional embodiments, the PTD may be a PTD peptide that isduplicated or multimerized. In certain embodiments, the PTD is one ormore of the TAT PTD peptide YARAAARQARA (SEQ ID NO: 14). In certainembodiments, the PTD is a multimer consisting of three of the TAT PTDpeptide YARAAARQARA (SEQ ID NO: 15). A HOXB4 protein that is fused orlinked to a multimeric PTD, such as, for example, a triplicatedsynthetic protein transduction domain (tPTD), may exhibit reducedlability and increased stability in cells. Such a HOXB4 construct mayalso be stable in serum-free medium and in the presence of hES cells.

Techniques for making fusion genes encoding fusion proteins are wellknown in the art. Essentially, the joining of various DNA fragmentscoding for different polypeptide sequences is performed in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed to generate a chimeric gene sequence (see, for example,Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley& Sons: 1992).

In certain embodiments, a fusion gene coding for a purification leadersequence, such as a poly-(His) sequence, may be linked to the N-terminusof the desired portion of the HOXB4 polypeptide or HOXB4-fusion protein,allowing the fusion protein be purified by affinity chromatography usinga Ni²⁺ metal resin. The purification leader sequence can then besubsequently removed by treatment with enterokinase to provide thepurified HOXB4 polypeptide (e.g., see Hochuli et al., (1987) J.Chromatography 411:177; and Janknecht et al., PNAS USA 88:8972).

In certain embodiments, a HOXB4 protein or functional variant or activedomain of it, is linked to the C-terminus or the N-terminus of a secondprotein or protein domain (e.g., a PTD) with or without an interveninglinker sequence. The exact length and sequence of the linker and itsorientation relative to the linked sequences may vary. The linker maycomprise, for example, 2, 10, 20, 30, or more amino acids and may beselected based on desired properties such as solubility, length, stericseparation, etc. In particular embodiments, the linker may comprise afunctional sequence useful for the purification, detection, ormodification, for example, of the fusion protein. In certainembodiments, the linker comprises a polypeptide of two or more glycines.

The protein domains and/or the linker by which the domains are fused maybe modified to alter the effectiveness, stability and/or functionalcharacteristics of HOXB4.

In certain embodiments, HOXB4 is ectopically expressed within thehemangioblast cell or is provided in the media. HOXB4 expressedectopically may be operably linked to a regulatory sequence. Regulatorysequences are art-recognized and are selected to direct expression ofthe HOXB4 polypeptide.

HOXB4 provided in the media may be excreted by another cell type. Theother cell type may be a feeder layer, such as a mouse stromal celllayer transduced to express excretable HOXB4. For example, HOXB4 may befused to or engineered to comprise a signal peptide, or a hydrophobicsequence that facilitates export and secretion of the protein.Alternatively, HOXB4, such as a fusion protein covalently ornon-covalently linked to a PTD, may be added directly to the media.Additionally, HOXB4 may be borne on a viral vector, such as a retroviralvector or an adenoviral vector. Such a vector could transduce either thehemangioblasts or other cells in their culture.

Depending on the HOXB4 protein used, in particular embodiments HOXB4 isadded to the media at selected times during the expansion of thehemangioblasts. Because the hemangioblasts are expanded in serum-freemedium, HOXB4 is relatively stable. Accordingly, in certain embodiments,a HOXB4 protein or fusion protein is added every day to the humanhemangioblasts. In other embodiments, a HOXB4 protein or fusion proteinis added every other day, and in still other embodiments, a HOXB4protein or fusion protein is added every 2 days. In one embodiment, aHOXB4 fusion protein, HOXB4-PTD, is added every 2 days to the media.

In certain embodiments, the hemangioblasts can be expanded in thepresence of any other growth factors or proteins that are present in anamount sufficient to expand such cells.

Hemangioblasts obtained from any source, including human or non-human EScells, bone marrow, placenta or umbilical cord blood, peripheral blood,or other tissue may be expanded according to the methods describedabove. Accordingly, in certain embodiments, a select number of purifiedhemangioblasts or enriched cells are mixed with serum-freemethylcellulose medium optimized for hemangioblast growth (e.g., BL-CFUmedium,). This medium may be supplemented with early stage cytokines(including, but not limited to, EPO, TPO, FL, VGF, BMPs like BMP2, BMP4and BMP7, but not BMP3) and HOXB4. In certain embodiments,erythropoietin (EPO) is added to the media. In certain embodiments, EPO,TPO and FL are added to the media. The cells are then plated ontoultra-low attachment culture dishes and grown in a CO₂ incubator. Asmentioned above, hemangioblast colonies exhibit a distinctive grape-likemorphology and are comparatively smaller than other cells and mayconsequently be distinguished from other cell types. The hemangioblastsmay also be tested for markers as well as for their ability todifferentiate further into either hematopoietic or endothelial celllineages. The hemangioblasts are subsequently isolated and expanded invitro. Media that may be used for expansion includes serum-freemethylcellulose medium optimized for hemangioblasts growth (e.g.,BL-CFU) supplemented with early stage cytokines and HOXB4. Early stagecytokines include, but are not limited to, EPO, TPO, FL, VEGF, BMPs likeBMP2, BMP4 and BMP7, but not BMP3. In certain embodiments,erythropoietin (EPO) is added to the medium. In further embodiments,EPO, TPO and FL are added to the medium.

Accordingly, a medium for expanding hemangioblasts may comprise VEGF,SCF, EPO, BMP-4, and HoxB4; in certain embodiments the medium mayfurther comprise TPO and FL. For example, single cells prepared from EBscultured for approximately 3.5 days, were collected and dissociated by0.05% trypsin-0.53 mM EDTA (Invitrogen) for 2-5 min, and a single cellsuspension was prepared by passing through 22 G needle 3-5 times. Cellswere collected by centrifugation at 1,000 rpm for 5 min. Cell pelletswere resuspended in 50-200 μl of Stemline I media. To expandhemangioblasts, single cell suspension derived from differentiation of 2to 5×10⁵ hES cells were mixed with 2 ml hemangioblast expansion media(HGM) containing 1.0% methylcellulose in Iscove's MDM, 1-2% Bovine serumalbumin, 0.1 mM 2-mercaptoethanol, 10 μg/ml rh-Insulin, 200 μg/ml ironsaturated human transferrin, 20 ng/ml rh-GM-CSF, 20 ng/ml rh-IL-3, 20ng/ml rh-IL-6, 20 ng/ml rh-G-CSF, 3 to 6 units/ml rh-Epo, 50 ng/mlrh-SCF, 50 ng/ml rh-VEGF and 50 ng/ml rh-BMP-4, and 1.5 μg/ml oftPTD-HoxB4, with/without 50 ng/ml of Tpo and FL. The cell mixtures wereplated on ultra-low dishes and incubated at 37° C. in 5% CO₂ for 4-6days.

In certain situations it may be desirable to obtain hemangioblasts froma patient or patient relative and expand said hemangioblasts in vitro.Such situations include, for example, a patient scheduled to beginchemotherapy or radiation therapy, or other situations wherein anautologous HSC transplantation (using the patient's own stem cells) maybe used. Thus, the present invention provides methods of treatingpatients in need of cell-based therapy (for example, patients in need ofhematopoietic reconstitution or treatment, or blood vessel growth ortreatment of vascular injuries including ischemia, see below) using theexpanded hemangioblasts or hemangioblast lineage cells of the invention,wherein the hemangioblasts are obtained from the bone marrow, blood, orother tissue of the patient or a patient relative. Accordingly, incertain embodiments, methods of treating a patient in need ofhemangioblasts (or hemangioblast lineage cells) may comprise a step ofisolating hemangioblasts from the patient or a patient relative.Hemangioblasts isolated from the patient or patient relative may beexpanded in vitro according to the methods of the present invention andsubsequently administered to the patient. Alternatively the expandedhemangioblasts may be grown further to give rise to hematopoietic cellsor endothelial cells before patient treatment.

It is also possible to obtain human ES cells from such a patient by anymethod known in the art, such as somatic cell nuclear transfer.Hemangioblasts of that patient may then be generated and expanded fromhis own ES cells using a method of this invention. Those hemangioblastsor lineage derivatives thereof may be administered to that patient or tohis relatives.

Using the methods of the present invention, human hemangioblasts areexpanded to reach commercially large quantities which can besubsequently used in various therapeutic and clinical applications.Furthermore, the hemangioblasts obtained by the methods disclosed hereinmay be differentiated further to give rise to either hematopoietic orendothelial cell lineages for use in clinical applications.

The hemangioblasts obtained from the method of this invention forgenerating and expanding human hemangioblasts from human ES cells havethe potential to differentiate into at least endothelial cells orhematopoietic cells (i.e., they are at least bi-potential). Otherhemangioblasts may be bi-potential as well. Yet other hemangioblasts maybe able to differentiate into cells other than hematopoietic andendothelial cells, i.e., they are multi- or pluri-potential).

Engineering MHC Genes in Human Embryonic Stem Cells to ObtainReduced-Complexity Hemangioblasts

The human embryonic stem cells used as the starting point for the methodof generating and expanding human hemangioblast cells of this inventionmay also be derived from a library of human embryonic stem cells, eachof which is hemizygous or homozygous for at least one MHC allele presentin a human population. In certain embodiments, each member of saidlibrary of stem cells is hemizygous or homozygous for a different set ofMHC alleles relative to the remaining members of the library. In certainembodiments, the library of stem cells is hemizygous or homozygous forall MHC alleles that are present in a human population. In the contextof this invention, stem cells that are homozygous for one or morehistocompatibility antigen genes include cells that are nullizygous forone or more (and in some embodiments, all) such genes. Nullizygous for agenetic locus means that the gene is null at that locus, i.e., bothalleles of that gene are deleted or inactivated. Stem cells that arenullizygous for all MHC genes may be produced by standard methods knownin the art, such as, for example, gene targeting and/or loss ofheterozygocity (LOH). See, for example, United States patentpublications US 20040091936, US 20030217374 and US 20030232430, and U.S.provisional application No. 60/729,173, the disclosures of all of whichare hereby incorporated by reference herein.

Accordingly, the present invention relates to methods of obtaininghemangioblasts, including a library of hemangioblasts, with reduced MHCcomplexity. Hemangioblasts and hemangioblast lineage cells with reducedMHC complexity will increase the supply of available cells fortherapeutic applications as it will eliminate the difficultiesassociated with patient matching. Such cells may be derived from stemcells that are engineered to be hemizygous or homozygous for genes ofthe MHC complex.

A human ES cell may comprise modifications to one of the alleles ofsister chromosomes in the cell's MHC complex. A variety of methods forgenerating gene modifications, such as gene targeting, may be used tomodify the genes in the MHC complex. Further, the modified alleles ofthe MHC complex in the cells may be subsequently engineered to behomozygous so that identical alleles are present on sister chromosomes.Methods such as loss of heterozygosity (LOH) may be utilized to engineercells to have homozygous alleles in the MHC complex. For example, one ormore genes in a set of MHC genes from a parental allele can be targetedto generate hemizygous cells. The other set of MHC genes can be removedby gene targeting or LOH to make a null line. This null line can be usedfurther as the embryonic cell line in which to drop arrays of the HLAgenes, or individual genes, to make a hemizygous or homozygous bank withan otherwise uniform genetic background.

In one aspect, a library of ES cell lines, wherein each member of thelibrary is homozygous for at least one HLA gene, is used to derivehemangioblasts according to the methods of the present invention. Inanother aspect, the invention provides a library of hemangioblasts(and/or hemangioblast lineage cells), wherein several lines of ES cellsare selected and differentiated into hemangioblasts. Thesehemangioblasts and/or hemangioblast lineage cells may be used for apatient in need of a cell-based therapy.

Accordingly, certain embodiments of this invention pertain to a methodof administering human hemangioblasts, hematopoietic stem cells, orhuman endothelial cells that have been derived from reduced-complexityembryonic stem cells to a patient in need thereof. In certainembodiments, this method comprises the steps of: (a) identifying apatient that needs treatment involving administering humanhemangioblasts, hematopoietic stem cells, or human endothelial cells tohim or her; (b) identifying MHC proteins expressed on the surface of thepatient's cells; (c) providing a library of human hemangioblasts ofreduced WIC complexity made by the method for generating and expandinghuman hemangioblast cells in vitro of the present invention; (d)selecting the human hemangioblast cells from the library that match thispatient's MHC proteins on his or her cells; (e) optionallydifferentiating the human hemangioblast cells identified in step (d)into human hematopoietic stem cells, endothelial cells or both, or cellsthat are further differentiated in either or both of these two lineages,depending on need; (f) administering any of the cells from step (d)and/or (e) to said patient. This method may be performed in a regionalcenter, such as, for example, a hospital, a clinic, a physician'soffice, and other health care facilities. Further, the hemangioblastsselected as a match for the patient, if stored in small cell numbers,may be expanded prior to patient treatment.

Human Hemangio-Colony Forming Cells/Hemangioblasts

In certain aspects, the present invention provides human hemangio-colonyforming cells. These cells are a unique, primitive cell type with avariety of therapeutic and other uses. Furthermore, this cell typeprovides an important tool for studying development of at least thehematopoietic and/or endothelial lineages. As such, the inventioncontemplates various preparations (including pharmaceuticalpreparations) and compositions comprising human hemangio-colony formingcells, as well as preparations (including pharmaceutical preparations)and compositions comprising one or more cell types partially orterminally differentiated from hemangio-colony forming cells.

Human hemangio-colony forming cells of the present invention have atleast one of the following structural characteristics: (a) candifferentiate to give rise to at least hematopoietic cell types orendothelial cell types; (b) can differentiate to give rise to at leasthematopoietic cell types and endothelial cell types; (c) are looselyadherent to each other (to other human hemangio-colony forming cells;(d) do not express CD34 protein; (e) do not express CD31 protein; (f) donot express KDR protein; (g) do not express CD133 protein; (h) expressGATA2 protein; (i) express LMO2 protein. In certain embodiments, humanhemangio-colony forming cells have at least two, at least three, atleast four, at least five, at least six, at least seven, at least eight,or at least nine of the structural or functional characteristicsdetailed herein.

The invention provides for human hemangio-colony forming cells. Suchcells can differentiate to produce at least hematopoietic and/orendothelial cell types. In certain embodiments, the cells arecharacterized as being loosely adherent to other human hemangio-colonyforming cells. Alternatively or additionally, these cells may also bedescribed based on expression or lack of expression of certain markers.For example, these cells may also be described based on lack ofexpression of at least one of the following proteins: CD34, KDR, CD133,and CD31.

As detailed above, one of the interesting properties of humanhemangio-colony forming cells is that they are loosely adherent to eachother. Because these cells are only loosely adherent to each other,cultures or colonies of hemangio-colony forming cells can be dissociatedto single cells using only mechanical dissociation techniques andwithout the need for enzymatic dissociation techniques. The cells aresufficiently loosely adherent to each other that mechanical dissociationalone, rather than enzymatic dissociation or a combination thereof, issufficient to disaggregate the cultures or colonies withoutsubstantially impairing the viability of the cells. In other words,mechanical dissociation does not require so much force as to causesubstantial cell injury or death.

This property is not only useful in describing the cells anddistinguishing them phenotypically from other cell types, but it alsohas significant therapeutic implications. For example, relatively largenumbers (greater than 1×10⁶ or even greater than 1×10⁷ or even greaterthan 1×10⁸) of the hemangio-colony forming cells can be injected intohumans or other animals with substantially less risk of causing clots oremboli, or otherwise lodging in the lung. This is a significant advancein cellular therapy. The ability to safely administer relatively largenumbers of cells makes cellular therapy practical and possible for theeffective treatment of an increasing number of diseases and conditions.

The term “loosely adherent” is described qualitatively above and refersto behavior of the human hemangio-colony forming cells with respect toeach other. Cultures or colonies of hemangio-colony forming cells can bedissociated to single cells using only mechanical dissociationtechniques and without the need for enzymatic dissociation techniques.The cells are sufficiently loosely adherent to each other thatmechanical dissociation alone, rather than enzymatic dissociation or acombination thereof, is sufficient to disaggregate the cultures orcolonies without substantially impairing the viability of the cells. Inother words, mechanical dissociation does not require so much force asto cause substantial cell injury or death.

The term can also be described more quantitatively. For example and incertain embodiments, the term “loosely adherent” is used to refer tocultures or colonies of hemangio-colony forming cells wherein at least50% of the cells in the culture can be dissociated to single cells usingonly mechanical dissociation techniques and without the need forenzymatic dissociation techniques. In other embodiments, the term refersto cultures in which at least 60%, 65%, 70%, or 75% of the cells in theculture can be dissociated to single cells using only mechanicaldissociation techniques and without the need for enzymatic dissociationtechniques. In still other embodiments, the term refers to cultures inwhich at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% ofthe cells in the culture can be dissociated to single cells using onlymechanical dissociation techniques and without the need for enzymaticdissociation techniques.

The ability to dissociate the hemangio-colony forming cells using onlymechanical dissociation techniques and without the need for enzymaticdissociation techniques can be further quantitated based on the healthand viability of the cells following mechanical dissociation. In otherwords, if dissociation without enzymatic techniques requires so muchmechanical force that a significant number of the cells are damaged orkilled, the cells are not loosely adherent, as defined herein. Forexample and in certain embodiments, the term “loosely adherent” refersto cultures of cells that can be dissociated to single cells using onlymechanical dissociation techniques and without the need for enzymaticdissociation techniques, without substantially impairing the health orviability or the cells in comparison to that observed when the samecells are dissociated using enzymatic dissociation techniques. Forexample, the health or viability of the cells is decreased by less than15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or even less than 1% incomparison to that observed when a culture of the same cells aredissociated using enzymatic dissociation techniques.

Exemplary enzymatic dissociation techniques include, but are not limitedto, treatment with trypsin, collagenase, or other enzymes that disruptcell-cell or cell-matrix interactions. Exemplary mechanical dissociationtechniques include, but are not limited to, one or more passages througha pipette.

Human hemangio-colony forming cells according to the present inventionare defined structurally and functionally. Such cells can be generatedfrom any of a number of sources including from embryonic tissue,prenatal tissue, perinatal tissue, and even from adult tissue. By way ofexample, human hemangio-colony forming cells can be generated from humanembryonic stem cells, other embryo-derived cells (blastocysts,blastomeres, ICMs, embryos, trophoblasts/trophectoderm cells,trophoblast stem cells, primordial germ cells, embryonic germ cells,etc.), amniotic fluid, amniotic stem cells, placenta, placental stemcells, and umbilical cord.

The invention provides human hemangio-colony forming cells, compositionscomprising human hemangio-colony forming cells, and preparations(including pharmaceutical preparations) comprising human hemangio-colonyforming cells. Certain features of these aspects of the invention aredescribed in detail below. The invention contemplates combinations ofany of the following aspects and embodiments of the invention.

In one aspect, the invention provides a human hemangio-colony formingcell. The cell can differentiate to produce at least hematopoieticand/or endothelial cell types. In certain embodiments, the cell isloosely adherent to other human hemangio-colony forming cells. Incertain embodiments, the cell does not express CD34 protein. In certainother embodiments, the cell does not express one or more of (e.g., thecell does not express at least one, at least two, at least three, or atleast four of the following proteins) the following proteins: CD34,CD31, CD133, KDR. In certain other embodiments, the cell does expressGATA2 and/or LMO2 protein.

In another aspect, the invention provides a human hemangio-colonyforming cell. The cell, which cell can differentiate to produce at leasthematopoietic and/or endothelial cell types, and the cell does notexpress any of the following proteins: CD34, CD31, KDR, and CD133. Incertain embodiments, the cell is loosely adherent to other humanhemangio-colony forming cells. In other embodiments, the cell doesexpress GATA2 and/or LMO2 protein.

In another aspect, the invention provides a cell culture comprising asubstantially purified population of human hemangio-colony formingcells. The cells can differentiate to produce at least hematopoietic andendothelial cell types, and the cells are loosely adherent to eachother. In certain embodiments, the cell does not express CD34 protein.In certain other embodiments, the cell does not express one or more of(e.g., the cell does not express at least one, at least two, at leastthree, or at least four of the following proteins) the followingproteins: CD34, CD31, CD133, KDR. In certain other embodiments, the celldoes express GATA2 and/or LMO2 protein.

In another aspect, the invention provides a cell culture comprisinghuman hemangio-colony forming cells differentiated from embryonictissue. In certain embodiments, the hemangio-colony forming cells areloosely adherent to each other. In certain embodiments, the cells candifferentiate to produce at least hematopoietic and/or endothelial celltypes, and the cells are loosely adherent to each other. In certainembodiments, the cell does not express CD34 protein. In certain otherembodiments, the cell does not express one or more of (e.g., the celldoes not express at least one, at least two, at least three, or at leastfour of the following proteins) the following proteins: CD34, CD31,CD133, KDR. In certain other embodiments, the cell does express GATA2and/or LMO2 protein.

In another aspect, the invention provides a cell culture comprisinghuman hemangio-colony forming cells, which cells can differentiate toproduce at least hematopoietic and/or endothelial cell types. In certainembodiments, the cells are loosely adherent to each other. In certainembodiments, the cell does not express CD34 protein. In certain otherembodiments, the cell does not express one or more of (e.g., the celldoes not express at least one, at least two, at least three, or at leastfour of the following proteins) the following proteins: CD34, CD31,CD133, KDR. In certain other embodiments, the cell does express GATA2and/or LMO2 protein.

In another aspect, the invention provides a pharmaceutical preparationcomprising human hemangio-colony forming cells, which cells candifferentiate to produce at least hematopoietic and/or endothelial celltypes. In certain embodiments, the hemangio-colony forming cells areloosely adherent to each other. In certain embodiments, the cell doesnot express CD34 protein. In certain other embodiments, the cell doesnot express one or more of (e.g., the cell does not express at leastone, at least two, at least three, or at least four of the followingproteins) the following proteins: CD34, CD31, CD133, KDR. In certainother embodiments, the cell does express GATA2 and/or LMO2 protein. Thepharmaceutical preparation can be prepared using any pharmaceuticallyacceptable carrier or excipient.

In another aspect, the invention provides a pharmaceutical preparationcomprising human hemangio-colony forming cells, wherein thehemangio-colony forming cells do not express any of the followingproteins: CD34, CD31, KDR, and CD133. In certain embodiments, thehemangio-colony forming cells can differentiate to produce at leasthematopoietic and/or endothelial cell types. In certain embodiments, thehemangio-colony forming cells are loosely adherent to each other. Incertain other embodiments, the cell does express GATA2 and/or LMO2protein. The pharmaceutical preparation can be prepared using anypharmaceutically acceptable carrier or excipient.

In certain embodiments of any of the foregoing, the composition orpharmaceutical preparation comprises at least 1×10⁵ humanhemangio-colony forming cells. In certain other embodiment, of any ofthe foregoing, the composition or pharmaceutical preparation comprisesat least 1×10⁶, at least 5×10⁶, at least 1×10⁷, or greater than 1×10⁷human hemangio-colony forming cells.

Additional cells, compositions, and preparations include cells partiallyor terminally differentiated from human hemangio-colony forming cells.For example, the invention contemplates compositions and preparationscomprising one or more hematopoietic and/or endothelial cell typedifferentiated from a hemangio-colony forming cell. Exemplaryhematopoietic cell types include hematopoietic stem cells, platelets,RBCs, lymphocytes, megakaryocytes, and the like. By way of furtherexamples, the invention contemplates compositions and preparationscomprising one or more other cell type, such as one or more partially orterminally differentiated mesodermal cell type, differentiated fromhemangio-colony forming cells.

In certain embodiments of any of the foregoing, the invention provides acryopreserved preparation of human hemangio-colony cells or cellspartially or terminally differentiated therefrom.

In certain embodiments of any of the foregoing, the invention providesfor the therapeutic use of human hemangio-colony forming cells, orcompositions or preparations of human hemangio-colony forming cells.Such cells and preparations can be used in the treatment of any of theconditions or diseases detailed throughout the specification, as well asin the blood banking industry. Furthermore, cells differentiated fromhuman hemangio-colony forming cells, or compositions or preparations ofhuman hemangio-colony forming cells, can be used therapeutically in thetreatment of any of the conditions or diseases detailed throughout thespecification, as well as in the blood banking industry.

The human hemangio-colony forming cells of the invention are can be usedtherapeutically. Additionally or alternatively, human hemangio-colonyforming cells can be used to study development of endothelial andhematopoietic lineages or in screening assays to identify factors thatcan be used, for example, to (i) maintain human hemangio-colony formingcells or (ii) to promote differentiation of human hemangio-colonyforming cells to one or more partially or terminally differentiated celltypes. Furthermore, human hemangio-colony forming cells can be used togenerate one or more partially or terminally differentiated cell typesfor in vitro or in vivo use.

The human hemangio-colony forming cells of the invention can be used inany of the methods or application described in the present applicationincluding, but not limited to, in the treatment of any of the diseasesor conditions described herein.

Cell Preparations Comprising Hemangioblasts Expanded In Vitro

In certain embodiments of the present invention, mammalian (includinghuman) hemangioblasts are expanded to reach commercial quantities andare used in various therapeutic and clinical applications. In particularembodiments, hemangioblasts are expanded to reach cell numbers on theorder of 10,000 to 4 million (or more). These cell numbers may bereached within 3-4 days of starting the initial preparations.Accordingly, the present invention relates to preparations comprisinglarge numbers of hemangioblasts, said preparations comprising at least10,000, 50,000, 100,000, 500,000, a million, 2 million, 3 million or 4million cells.

This invention also provides for a solution, a composition, and apreparation comprising large numbers of hemangioblasts, said solution,said composition, and said preparation comprising at least 10,000,50,000, 100,000, 500,000, a million, 2 million, 3 million or 4 millioncells. The hemangioblasts could be human.

Other aspects of the present invention relate to differentiating thehemangioblasts obtained by the methods disclosed herein into eitherhematopoietic or endothelial cell lineages, or both, that aresubsequently used in clinical applications. Thus, the present inventionalso relates to cell preparations comprising large numbers ofhematopoietic or endothelial cells. The invention also relates todifferentiating the hemangioblasts obtained by the methods disclosedherein into other cell lineages, other than hematopoietic andendothelial cells. Thus, the present invention also relates to cellpreparations comprising large numbers of other hemangioblast-derivedcells.

Compositions and preparations comprising large numbers (e.g., thousandsor millions) of hemangioblasts may be obtained by expandinghemangioblasts that are obtained as described above. Accordingly, theinvention pertains to compositions and preparations comprising largenumbers of hemangioblasts achieved by expanding ES cells (such as humanES cells) or hemangioblasts obtained from cord blood, peripheral bloodor bone marrow. Further, as the methods of expansion may be applied tohemangioblasts of mouse, rat, bovine, or non-human primate origin, forexample, the present invention also relates to compositions andpreparations comprising large numbers of hemangioblasts of other speciesin addition to human. The hemangioblasts to be expanded by the methodsof this invention may be bi-potential, i.e., can differentiate intoeither endothelial cells or hematopoietic stem cells. In certainembodiments, the human hemangioblasts generated and expanded from humanES cells are bi-potential. Hemangio-colony forming cells are capable ofdifferentiating to give rise to at least hematopoietic cell types orendothelial cell types. Hemangio-colony forming cells are preferablybi-potential and capable of differentiating to give rise to at leasthematopoietic cell types and endothelial cell types. As such,hemangio-colony forming cells of the present invention are at leastuni-potential, and preferably bi-potential. Additionally however,hemangio-colony forming cells may have a greater degree of developmentalpotential and can, in certain embodiments, differentiate to give rise tocell types of other lineages. In certain embodiments, thehemangio-colony forming cells are capable of differentiating to giverise to other mesodermal derivatives such as cardiac cells (for example,cardiomyocytes) and/or smooth muscle cells.

Mammalian Hemangioblast Cell Markers

As described above, the hemangio-colony forming cells lack certainfeatures characteristic of mature endothelial or hematopoietic cells.These hemangio-colony forming cells or hemangioblasts, however, may beidentified by various markers such as, for example, CD71+, GATA-1 andGATA-2 proteins, CXCR-4, and TPO and EPO receptors. In additionalembodiments, the hemangioblasts express LMO-2. Hemangioblasts mayadditionally be characterized by the absence or low expression of othermarkers. Accordingly, hemangioblasts may be CD34− CD31−, and KDR−. Infurther embodiments, the hemangioblasts may be CD34−, CD31−, KDR−, andCD133−.

Accordingly, in certain embodiments, the hemangioblasts generated andexpanded by the methods of present invention are characterized by thepresence or absence of any one or more of the markers listed in Table 2of WO2007/120811, incorporated herein by reference in its entirety. Forexample, the hemangioblasts may test negative for expression of any oneor more of the markers listed in Table 2 that is denoted as “−” under“BL-CFC”. Accordingly, in some embodiments, the hemangioblasts may benegative for CD34 expression. The cells may additionally oralternatively be negative for CD31, CD133, and/or KDR expression. Infurther embodiments, the hemangioblasts may express any of the markersdenoted in Table 2 with “+”. For example, the cells may express one ormore of the markers LMO-2 and GATA-2. Expression of a marker may beassessed by any method, such as, for example, immunohistochemistry orimmunoblotting to test for protein expression, or mRNA analysis to testfor expression at the RNA level.

Deriving Hemangioblast Lineage Cells

The methods and cell preparations of the present invention also relateto hemangioblast derivative cells. Human hemangioblasts generated andexpanded by this invention and mammalian hemangioblasts expanded by themethods of the invention may be differentiated in vitro to obtainhematopoietic cells (including hematopoietic stem cells (HSCs)) orendothelial cells, as well as cells that are further differentiated inthese two lineages. These cells may subsequently be used in thetherapeutic and commercial applications described below.

In certain embodiments, hematopoietic cells are derived by growing thehemangioblasts in serum-free BL-CFU for 3-10 days. In other embodiments,single-cell suspensions of hES-derived BL-CFC cells are grown for 10-14days. Maintaining serum-free conditions is optimal insofar as serum-freeconditions facilitate scale-up production and compliance with regulatoryguidelines as well as reduce cost. Hemangioblasts of the presentinvention may also be grown in serum-free Hem-culture (Bhatia et al.1997 J Exp Med (186): 619-624), which sustains human hematopoietic stemcells and comprises BSA (e.g., 1% BSA), insulin (e.g., 5 μg/ml humaninsulin), transferrin media or transferrin (e.g., 100 μg/ml humantransferrin), L-glutamine, beta-mercaptoethanol (e.g., 10⁻⁴ M), andgrowth factors. The growth factors may comprise SCF (e.g., 300 ng/ml),granulocytic-colony-stimulating factor (G-CSF) (e.g., 50 ng/ml), Flt-3(e.g., 300 ng/ml), IL-3 (e.g., 10 ng/ml), and IL-6 (e.g., 10 ng/ml).Other factors useful for obtaining hematopoietic cells fromhemangioblasts include thrombopoietin (TPO) and VEGF (see, for example,Wang et al. 2005 Ann NY Acad Sci (1044): 29-40) and BMP-4. Thehemangioblasts may also be grown in serum-free methylcellulose mediumsupplemented with a multilineage hematopoietic growth factor cocktail.Thus, the hemangioblasts may be grown in methylcellulose in Iscovemodified Dulbecco medium (IMDM) comprising BSA, saturated humantransferrin, human LDL, supplemented with early acting growth factors(e.g., c-kit ligand, flt3 ligand), multilineage growth factors (e.g.,IL-3, granulocyte macrophage-CSF (GM-CSF)), and unilineage growthfactors (e.g., G-CSF, M-CSF, EPO, TPO)), VEGF, and bFGF. Alternatively,the hemangioblasts may be grown in medium comprising unilineage growthfactors to support the growth of one type of hematopoietic cell (e.g.,red blood cells, macrophages, or granulocytes).

In one embodiment, hemangioblast colonies are resuspended in Stemline Imedia. Cells are then mixed with 1 ml of serum-free hematopoietic CFUmedia (H4436, Stem Cell Technologies™) plus 1.5 μg/ml of tPTD-HoxB4 and0.5% EX-CYTE (Serologicals Proteins Inc.™). The cell mixtures are thenplated on cell culture untreated plates and incubated at 37° C. for10-14 days. Hematopoietic CFUs arising following 10-14 days afterinitial plating may be characterized morphologically, such as bystaining with Wright-Giemsa dye.

Hematopoietic cells may also be derived from the hemangioblast usingother conditions known in the art (e.g., in media comprising IMDM, 30%fetal calf serum (FCS), 1% bovine serum albumin (BSA), 10⁻⁴ Mbeta-mercaptoethanol, and 2 mM L-glutamine). Further, in otherembodiments basic fibroblast growth factor may be used to promote bothBL-CFC frequency within EBs and promote hematopoietic differentiation(Faloon et al. 2000 Development (127): 1931-1941). In yet otherembodiments, the growth factor hemangiopoietin (HAPO) is used to promotegrowth and hematopoietic differentiation of the hemangioblasts (Liu etal. 2004 Blood (103): 4449-4456). The differentiation into hematopoieticcells may be assessed by CD45 status (CD45+) and the CFU assay, forexample.

To form hematopoietic cells, human hemangioblasts may be grown for 3-10days, or optionally for longer periods of time (e.g., 10-14 days) inCFU-medium. Human hemangioblasts of the present invention are able toform CFUs comprising granulocytes, erythrocytes, macrophages, andmegakaryocytes (CFU-GEMM/mix) as well as colony forming units containingonly one of the latter cell types (e.g., CFU-G, CFU-E, CFU-M, andCFU-GM). In certain embodiments, single-cell suspensions of hES-derivedBL-CFC cells are grown for 10-14 days to derive hematopoietic cells suchas, for example, erythroid, myeloid, macrophage, and multilineagehematopoietic cells.

Other aspects of the invention relate to endothelial cells derived fromthe human hemangioblasts obtained and expanded or mammalianhemangioblasts expanded by the methods described herein. Thehemangioblasts may be grown in conditions favorable to endothelialmaturation.

In certain embodiments of the present invention, to obtain endothelialcells, hemangioblasts are first plated onto a fibronectin-coated surfaceand following 3-5 days (or in other embodiments 3-7 days), are replatedonto a thick layer of Matrigel to support differentiation intoendothelial cells. These conditions maintain the serum-free conditionsestablished during hemangioblast development. Alternatively,hemangioblasts may be grown in media known to support differentiationinto endothelial cells. Such conditions include, for example,Endo-culture comprising 20% fetal bovine serum (FBS), 50 ng/mlendothelial cell growth supplement (i.e., pituitary extracts), 10 IU/mlheparin, and 5 ng/ml human VEGF-A₁₆₅ (Terramani et al. 2000 In vitroCell Dev Biol Anim (36): 125-132). Other conditions known in the artinclude medium supplemented with 25% FCS/horse serum, and in someembodiments heparin (e.g., 10 U/ml), insulin like growth factor (IGF1)(e.g., 2 ng), and EC growth supplement (EGGS, e.g., 100 μg). The growthfactors VEGF and EGF may also be used in combination with HAPO tosupport endothelial differentiation (Liu et al. 2004). Thehemangioblasts may also be seeded onto dishes coated with collagen andfibronectin, for example, to promote differentiation into endothelialcells. Cells may be analyzed for von Willebrand factor (vWF) andendothelial nitric oxide synthase (eNOS) and the ability to form anendothelial network in vitro.

Accordingly, to form endothelial cells, hemangioblast colonies derivedby the methods described above are picked and replated ontofibronectin-coated culture plates optimized for the first step towardsendothelial differentiation. The cells may be plated in EGM-2 or EGM-2MVcomplete media (Cambrex™). Following 3 to 5 days, and in alternativeembodiments 3 to 7 days, the cells are re-plated on a surface thatsupports endothelial differentiation, such as on a layer of Matrigel.Following 16-24 hours of incubation, the formation of branchedtube-cords suggests typical endothelial cell behavior.Endothelial-specific assays such as LDL-uptake may also be used toconfirm that these cells are of endothelial nature.

In other aspects of the invention, human hemangioblasts generated andexpanded by this invention and mammalian hemangioblasts expanded by themethods of the invention may be differentiated in vitro to obtain othercells, as well as cells that are further differentiated from these celllineages. Such additional cell lineages may be derived from thehemangioblasts generated and expanded by this invention and mammalianhemangioblasts expanded by the methods of the invention because thehemangioblast cells may have an even greater degree of developmentalpotential beyond differentiating into hematopoietic and endothelialcells.

Non-Engrafting Hemangio Cells

The present invention provides a novel cell population that shares somecharacteristics of previously identified hemangioblasts andhemangio-colony forming cells. However, the novel cell populationdescribed herein is distinct in that it does not engraft into the bonemarrow when administered to immunodeficient animals. This novelprogenitor cell population is useful for the study of basicdevelopmental and stem cell biology, is useful to generate partially andterminally differentiated cell type in vitro and in vivo, and is usefulfor the development of therapeutics. Additionally, these cells can beused in screening assays to identify, for example, (i) factors orconditions that promote the expansion of non-engrafting hemangio cellsand (ii) factors or conditions that promote the generation of one ormore differentiated cell type from non-engrafting hemangio cells.Identified factors and conditions can be used in the production ofcell-based and cell free therapies, in the production of mediums andformulations, and in the study of developmental and stem cell biology.

Overview

The present invention provides non-engrafting hemangio cells,compositions and preparations comprising non-engrafting hemangio cells,methods of producing and expanding non-engrafting hemangio cells,methods of producing differentiated cell types from non-engraftinghemangio cells, and methods of using non-engrafting hemangio cells orcells derived there from therapeutically.

The methods described herein can be used to generate humannon-engrafting hemangio cells. However, cells can be obtained from otherspecies including, but not limited to, mice, rats, rabbits, cows, dogs,cats, sheep, pigs, and non-human primates.

This invention provides a method for expanding mammalian non-engraftinghemangio cells obtained from any source, including ES cells, blastocystsor blastomeres, cord blood from placenta or umbilical tissue, peripheralblood, bone marrow, or other tissue or by any other means known in theart. In certain embodiments, human non-engrafting hemangio cells aregenerated from embryonic stem cells or other pluripotent stem cells. Byway of example, human non-engrafting hemangio cells can be generatedfrom embryonic stem cells, as well as from iPS cells. In otherembodiments, non-engrafting hemangio cells are generated from humanembryo-derived cells. Human embryo-derived cells may be a substantiallyhomogeneous population of cells, a heterogeneous population of cells, orall or a portion of an embryonic tissue. As an example of embryo-derivedcells that can be used in the methods of the present invention, humannon-engrafting hemangio cells can be generated from human embryonic stemcells. Such embryonic stem cells include embryonic stem cells derivedfrom or using, for example, blastocysts, plated ICMs, one or moreblastomeres, or other portions of a pre-implantation-stage embryo orembryo-like structure, regardless of whether produced by fertilization,somatic cell nuclear transfer (SCNT), parthenogenesis, androgenesis, orother sexual or asexual means. In certain embodiments, non-engraftinghemangio cells are generated from pluripotent stem cells. Exemplarypluripotent stem cells include, but are not limited to, embryonic stemcells and iPS cells. In certain embodiments, human non-engraftinghemangio cells are generated from non-pluripotent cells. Non-pluripotentcells may include somatic cells, such as cells derived from skin, bone,blood, connective tissue, heart, kidney, lung, liver, or any otherinternal organ. In certain embodiments, the non-pluripotent cells may becells derived from connective tissue, such as fibroblasts. In certainembodiments, the non-pluripotent cells are cells derived from an adulttissue.

In certain embodiments, non-engrafting hemangio cells can be furtherdifferentiated to hematopoietic stem cells and/or hematopoietic celltypes including, but not limited to, platelets and red blood cells. Suchcells may be used in transfusions or in other therapies. Although suchcells have numerous uses, a particularly important use would be inimproving the availability of blood for transfusions. In certainembodiments, the invention provides red blood cells differentiated fromnon-engrafting hemangio cells. Such differentiated red blood cells couldbe used for transfusions.

Further aspects of the invention relate to methods of generatingdifferentiated hematopoietic cells from non-engrafting hemangio cellsfor use in blood transfusions for those in need thereof. In certainembodiments, differentiated hematopoietic cells are transfused to treattrauma, blood loss during surgery, blood diseases such as anemia, Sicklecell anemia, or hemolytic diseases, or malignant disease. In certainembodiments, red blood cells are transfused to treat trauma, blood lossduring surgery, or blood diseases such as anemia, Sickle cell anemia, orhemolytic disease. In certain embodiments, a mixed population of redblood cells is transfused. It should be noted that many differentiatedhematopoietic cell types, particularly red blood cells, typically existin vivo as a mixed population. Specifically, circulating red blood cellsof varying levels of age and differentiation are found in vivo.Additionally, red blood cells mature over time so as to express lessfetal hemoglobin and more adult hemoglobin. The present inventioncontemplates transfusion of either purified populations of red bloodcells or of a mixed population of red blood cells having varying levelsof age and levels of differentiation. In particular embodiments, theinvention contemplates transfusion of red blood cells expressing fetalhemoglobin (hemoglobin F). Transfusion of red blood cells that expressfetal hemoglobin may be especially useful in the treatment of Sicklecell anemia. The ability to generate large numbers of cells fortransfusion will alleviate the chronic shortage of blood experienced inblood banks and hospitals across the country.

In certain embodiments, the methods of the invention allow for theproduction of universal cells for transfusion. Specifically, red bloodcells that are type O and Rh— can be readily generated and will serve asa universal blood source for transfusion. In certain embodiments, thered blood cells produced from the methods of the application arefunctional. In certain embodiments, the red blood cells expresshemoglobin F prior to transfusion. In certain embodiments, the red bloodcells carry oxygen. In certain embodiments, the red blood cells have alifespan equal to naturally derived red blood cells. In certainembodiments, the red blood cells have a lifespan that is 75% of that ofnaturally derived red blood cells. In certain embodiments, the red bloodcells have a lifespan that is 50% of that of naturally derived red bloodcells. In certain embodiments, the red blood cells have a lifespan thatis 25% of that of naturally derived red blood cells.

In certain embodiments, non-engrafting hemangio cells may have a greaterdevelopmental potential, and may differentiate to produce endothelialcell types, smooth muscle cell types, or cardiac cell types.

The methods of this invention allow for the in vitro expansion ofnon-engrafting hemangio cells to large quantities useful for a varietyof commercial and clinical applications. Expansion of non-engraftinghemangio cells in vitro refers to the proliferation of non-engraftinghemangio cells. While the methods of the invention enable the expansionof human non-engrafting hemangio cells to reach commercially usefulquantities, the present invention also relates to large numbers ofnon-engrafting hemangio cells and to cell preparations comprising largenumbers of human non-engrafting hemangio cells (for example, at least10,000, 100,000, or 500,000 cells). In certain embodiments, the cellpreparations comprise at least 1×10⁶ cells. In other embodiments, thecell preparations comprise at least 2×10⁶ human non-engrafting hemangiocells and in further embodiments at least 3×10⁶ human non-engraftinghemangio cells. In still other embodiments, the cell preparationscomprise at least 4×10⁶ human non-engrafting hemangio cells. Note thatthese cell preparations may be purified or substantially purified.However, in certain embodiments, suitable cell preparations comprise amixture of non-engrafting hemangio cells and hemangio-colony formingcells. The mixture may be any ratio, including mixtures comprising agreater proportion of non-engrafting hemangio cells and mixturescomprising a greater proportion of hemangio-colony forming cells.

The present invention relates to a solution, a preparation, or acomposition comprising between 10,000 and 4 million or more mammalian(such as human) non-engrafting hemangio cells. The number ofnon-engrafting hemangio cells in such a solution, a preparation, and acomposition may be any number between the range of 10,000 to 4 million,or more. This number could be, for example, 20,000, 50,000, 100,000,500,000, 1 million, etc.

Similarly, the invention relates to preparations of human non-engraftinghemangio progeny cells (e.g., human hematopoietic cells including humanhematopoietic stem cells). The invention further relates to methods ofproducing, storing, and distributing non-engrafting hemangio cellsand/or non-engrafting hemangio cell progeny.

The invention also provides methods and solutions suitable fortransfusion into human or animal patients. In particular embodiments,the invention provides methods of making red blood cells and/orplatelets, and/or other hematopoietic cell types for transfusion. Incertain embodiments, the invention is suitable for use in blood banksand hospitals to provide blood for transfusion following trauma, or inthe treatment of a blood-related disease or disorder. In certainembodiments, the invention provides red blood cells that are universaldonor cells. In certain embodiments, the red blood cells are functionaland express hemoglobin F prior to transfusion.

The invention also provides for human non-engrafting hemangio cells,cell cultures comprising a substantially purified population of humannon-engrafting hemangio cells, pharmaceutical preparations comprisinghuman non-engrafting hemangio cells and cryopreserved preparations ofthe non-engrafting hemangio cells. In certain embodiments, the inventionprovides for the use of the human non-engrafting hemangio cells in themanufacture of a medicament to treat a condition in a patient in needthereof. Alternatively, the invention provides the use of the cellcultures in the manufacture of a medicament to treat a condition in apatient in need thereof. The invention also provides the use of thepharmaceutical preparations in the manufacture of a medicament to treata condition in a patient in need thereof.

The non-engrafting hemangio cells can be identified and characterizedbased on their structural properties and/or function properties. Theseprogenitor cells do not engraft when administered to an immunodeficienthost. In certain embodiments, these cells are unique in that they areonly loosely adherent to each other (loosely adherent to othernon-engrafting hemangio cells). In embodiments in which the cells areloosely adherent, cultures or colonies of non-engrafting hemangio cellscan be dissociated to single cells using only mechanical dissociationtechniques and without the need for enzymatic dissociation techniques.In certain embodiments, the cells are sufficiently loosely adherent toeach other that mechanical dissociation alone, rather than enzymaticdissociation or a combination of mechanical and enzymatic dissociation,is sufficient to disaggregate the cultures or colonies withoutsubstantially impairing the viability of the cells. In other words,mechanical dissociation does not require so much force as to causesubstantial cell injury or death when compared to that observedsubsequent to enzymatic dissociation of cell aggregates.

In certain embodiments, the non-engrafting hemangio cells can be furtheridentified or characterized based on the expression or lack ofexpression (as assessed at the level of the gene or the level of theprotein) of one or more markers. In certain embodiments, thenon-engrafting hemangio cells have one or more of the characteristics ofhuman hemangio-colony forming cells. For example, in certainembodiments, non-engrafting hemangio cells can be identified orcharacterized based on lack of expression of one or more (e.g., thecells can be characterized based on lack of expression of at least one,at least two, at least three or at least four of the following markers)of the following cell surface markers: CD34, KDR, CD133, or CD31.Additionally or alternatively, non-engrafting hemangio cells can beidentified or characterized based on expression of GATA2 and/or LMO2.

Human Non-Engrafting Hemangio Cells

In certain aspects, the present invention provides human non-engraftinghemangio cells. These cells are a unique, primitive cell type with avariety of therapeutic and other uses. Furthermore, this cell typeprovides an important tool for studying development of at least thehematopoietic lineages. As such, the invention contemplates variouspreparations (including pharmaceutical preparations) and compositionscomprising human non-engrafting hemangio cells, as well as preparations(including pharmaceutical preparations) and compositions comprising oneor more cell types partially or terminally differentiated fromnon-engrafting hemangio cells. Without being bound by any particulartheory, these cells represent a distinct, somewhat more committed (thanhemangio-colony forming cells) stem cell population that retain theability to generate numerous hematopoietic cell types.

Non-engrafting hemangio cells of the present invention can be identifiedor characterized based on one or any combination of the structural orfunctional characteristics described for hemangio-colony forming cells.Note that although these cells can be derived from any of a number ofsources, for example, embryonic tissue, prenatal tissue, or perinataltissue, the term “non-engrafting hemangio cells” applies to cells,regardless of source, that do not engraft and that are capable ofdifferentiating to give rise to at least one hematopoietic cell type,and optionally have one or more of the foregoing structural orfunctional properties.

To illustrate, human non-engrafting hemangio cells of the presentinvention do not engraft when administered to a immunodeficient host andhave at least one of the following structural characteristics: (a) candifferentiate to give rise to at least one hematopoietic cell type; (b)can differentiate to give rise to at least hematopoietic cell types andendothelial cell types; (c) are loosely adherent to each other (to othernon-engrafting hemangio cells); (d) do not express CD34 protein; (e) donot express CD31 protein; (f) do not express KDR protein; (g) do notexpress CD133 protein; (h) express GATA2 protein; (i) express LMO2protein. In certain embodiments, human non-engrafting hemangio cellshave at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, or at least nine of thestructural or functional characteristics detailed herein.

As detailed above, one of the interesting properties of humannon-engrafting hemangio cells is that they are loosely adherent to eachother. Because these cells are only loosely adherent to each other,cultures or colonies of non-engrafting hemangio cells can be dissociatedto single cells using only mechanical dissociation techniques andwithout the need for enzymatic dissociation techniques. The cells aresufficiently loosely adherent to each other that mechanical dissociationalone, rather than enzymatic dissociation or a combination thereof, issufficient to disaggregate the cultures or colonies withoutsubstantially impairing the viability of the cells. In other words,mechanical dissociation does not require so much force as to causesubstantial cell injury or death.

This property is not only useful in describing the cells anddistinguishing them phenotypically from other cell types, but it alsohas significant therapeutic implications. For example, relatively largenumbers (greater than 1×10⁸ or even greater than 1×10⁷ or even greaterthan 1×10⁸) of the non-engrafting hemangio cells can be injected intohumans or other animals with substantially less risk of causing clots oremboli, or otherwise lodging in the lung. This is a significant advancein cellular therapy. The ability to safely administer relatively largenumbers of cells makes cellular therapy practical and possible for theeffective treatment of an increasing number of diseases and conditions.

The term “loosely adherent” is described qualitatively above and refersto behavior of the human non-engrafting hemangio cells with respect toeach other. Cultures or colonies of non-engrafting hemangio cells can bedissociated to single cells using only mechanical dissociationtechniques and without the need for enzymatic dissociation techniques.The cells are sufficiently loosely adherent to each other thatmechanical dissociation alone, rather than enzymatic dissociation or acombination thereof, is sufficient to disaggregate the cultures orcolonies without substantially impairing the viability of the cells. Inother words, mechanical dissociation does not require so much force asto cause substantial cell injury or death.

The term can also be described more quantitatively. For example and incertain embodiments, the term “loosely adherent” is used to refer tocultures or colonies of non-engrafting hemangio cells wherein at least50% of the cells in the culture can be dissociated to single cells usingonly mechanical dissociation techniques and without the need forenzymatic dissociation techniques. In other embodiments, the term refersto cultures in which at least 60%, 65%, 70%, or 75% of the cells in theculture can be dissociated to single cells using only mechanicaldissociation techniques and without the need for enzymatic dissociationtechniques. In still other embodiments, the term refers to cultures inwhich at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% ofthe cells in the culture can be dissociated to single cells using onlymechanical dissociation techniques and without the need for enzymaticdissociation techniques.

The ability to dissociate the non-engrafting hemangio cells using onlymechanical dissociation techniques and without the need for enzymaticdissociation techniques can be further quantitated based on the healthand viability of the cells following mechanical dissociation. In otherwords, if dissociation without enzymatic techniques requires so muchmechanical force that a significant number of the cells are damaged orkilled, the cells are not loosely adherent, as defined herein. Forexample and in certain embodiments, the term “loosely adherent” refersto cultures of cells that can be dissociated to single cells using onlymechanical dissociation techniques and without the need for enzymaticdissociation techniques, without substantially impairing the health orviability or the cells in comparison to that observed when the samecells are dissociated using enzymatic dissociation techniques. Forexample, the health or viability of the cells is decreased by less than15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% , 2%, or even less than 1% incomparison to that observed when a culture of the same cells aredissociated using enzymatic dissociation techniques.

Exemplary enzymatic dissociation techniques include, but are not limitedto, treatment with trypsin, collagenase, or other enzymes that disruptcell-cell or cell-matrix interactions. Exemplary mechanical dissociationtechniques include, but are not limited to, one or more passages througha pipette.

Human non-engrafting hemangio cells according to the present inventionare defined structurally and functionally. Such cells can be generatedfrom any of a number of sources including from embryonic tissue,prenatal tissue, perinatal tissue, and even from adult tissue. By way ofexample, human non-engrafting hemangio cells can be generated from humanembryonic stem cells, other embryo-derived cells (blastocysts,blastomeres, ICMs, embryos, trophoblasts/trophectoderm cells,trophoblast stem cells, primordial germ cells, embryonic germ cells,etc.), amniotic fluid, amniotic stem cells, placenta, placental stemcells, and umbilical cord. More generally, non-engrafting hemangio cellscan be generated from pluripotent cells, such as embryonic stem cells orpluripotent stem cells. Exemplary pluripotent stem cells include, butare not limited to, embryonic stem cells and induced pluripotent stemcells (iPS cells). Human non-engrafting hemangio cells can also begenerated from non-pluripotent cells, such as somatic cells, includingbut not limited to, cells derived from skin, bone, blood, connectivetissue, heart, kidney, lung, liver, or any other internal organ. Incertain embodiments, the non-pluripotent cells may be cells derived fromconnective tissue, such as fibroblasts. In certain embodiments, thenon-pluripotent cells are cells derived from an adult tissue.

The invention provides non-engrafting hemangio cells (such as humancells), compositions comprising human non-engrafting hemangio cells, andpreparations (including pharmaceutical preparations) comprising humannon-engrafting hemangio cells. Certain features of these aspects of theinvention are described in detail below. The invention contemplatescombinations of any of the following aspects and embodiments of theinvention, as well as combinations with the disclosure provided at U.S.application Ser. No. 11/787,262, which is incorporated by reference inits entirety.

As detailed above, hemangio-colony forming cells and/or non-engraftinghemangio cells can be produced from a variety of cells including, butnot limited to, pluripotent cells (embryonic stem cells, embryo-derivedcells, and induced pluripotent stem cells).

In one aspect, the invention provides a non-engrafting hemangio cells(such as human cells). The cell can differentiate to produce at leastone hematopoietic cell types. In certain embodiments, the cell isloosely adherent to other human non-engrafting hemangio cells. Incertain embodiments, the cell does not express CD34 protein. In certainother embodiments, the cell does not express one or more of (e.g., thecell does not express at least one, at least two, at least three, or atleast four of the following proteins) the following proteins: CD34,CD31, CD133, KDR. In certain other embodiments, the cell does expressGATA2 and/or LMO2 protein. In certain other embodiments, the cell sharesone or more than one (2, 3, 4, 5, 6, 7, 8, 9, 10) of the functional orstructural characteristics of human hemangio colony forming cells.

In another aspect, the invention provides a cell culture comprising asubstantially purified population of non-engrafting hemangio cells (suchas human cells). The cells can differentiate to produce at leasthematopoietic cell types. In certain embodiments, the cells are looselyadherent to each other. In certain embodiments, the cell does notexpress CD34 protein. In certain other embodiments, the cell does notexpress one or more of (e.g., the cell does not express at least one, atleast two, at least three, or at least four of the following proteins)the following proteins: CD34, CD31, CD133, KDR. In certain otherembodiments, the cell does express GATA2 and/or LMO2 protein. In certainother embodiments, the cell shares one or more than one (2, 3, 4, 5, 6,7, 8, 9, 10) of the functional or structural characteristics of humanhemangio colony forming cells.

In another aspect, the invention provides a cell culture comprisingnon-engrafting hemangio cells differentiated from embryonic tissue. Incertain embodiments, the invention provides a cell culture comprisingnon-engrafting hemangio cells differentiated from pluripotent cells(pluripotent stem cells). In certain embodiments, the non-engraftinghemangio cells are loosely adherent to each other. In certainembodiments, the cells can differentiate to produce at leasthematopoietic cell types, and the cells are loosely adherent to eachother. In certain embodiments, the cell does not express CD34 protein.In certain other embodiments, the cell does not express one or more of(e.g., the cell does not express at least one, at least two, at leastthree, or at least four of the following proteins) the followingproteins: CD34, CD31, CD133, KDR. In certain other embodiments, the celldoes express GATA2 and/or LMO2 protein. In certain other embodiments,the cell shares one or more than one (2, 3, 4, 5, 6, 7, 8, 9, 10) of thefunctional or structural characteristics of human hemangio colonyforming cells.

In another aspect, the invention provides a cell culture comprisinghuman non-engrafting hemangio cells, which cells can differentiate toproduce at least hematopoietic cell types. In certain embodiments, thecells are loosely adherent to each other. In certain embodiments, thecell does not express CD34 protein. In certain other embodiments, thecell does not express one or more of (e.g., the cell does not express atleast one, at least two, at least three, or at least four of thefollowing proteins) the following proteins: CD34, CD31, CD133, KDR. Incertain other embodiments, the cell does express GATA2 and/or LMO2protein. In certain other embodiments, the cell shares one or more thanone (2, 3, 4, 5, 6, 7, 8, 9, 10) of the functional or structuralcharacteristics of human hemangio colony forming cells.

In another aspect, the invention provides a pharmaceutical preparationcomprising human non-engrafting hemangio cells, which cells candifferentiate to produce at least hematopoietic cell types. In certainembodiments, the non-engrafting hemangio cells are loosely adherent toeach other. In certain embodiments, the cell does not express CD34protein. In certain other embodiments, the cell does not express one ormore of (e.g., the cell does not express at least one, at least two, atleast three, or at least four of the following proteins) the followingproteins: CD34, CD31, CD133, KDR. In certain other embodiments, the celldoes express GATA2 and/or LMO2 protein. In certain other embodiments,the cell shares one or more than one (2, 3, 4, 5, 6, 7, 8, 9, 10) of thefunctional or structural characteristics of human hemangio colonyforming cells. The pharmaceutical preparation can be prepared using anypharmaceutically acceptable carrier or excipient.

In another aspect, the invention provides a pharmaceutical preparationcomprising human non-engrafting hemangio cells. The pharmaceuticalpreparation can be prepared using any pharmaceutically acceptablecarrier or excipient.

In certain embodiments of any of the foregoing, the composition orpharmaceutical preparation comprises at least 1×10⁵ human non-engraftinghemangio cells. In certain other embodiment, of any of the foregoing,the composition or pharmaceutical preparation comprises at least 1×10⁶,at least 5×10⁶, at least 1×10⁷, or greater than 1×10⁷ humannon-engrafting hemangio cells. In certain embodiments, the preparationis a purified or substantially purified preparation. In otherembodiments, the preparation comprises a mixture of non-engraftinghemangio cells and other cell types. For example, a mixture ofnon-engrafting hemangio cells and hemangio-colony forming cells.

Additional cells, compositions, and preparations include cells partiallyor terminally differentiated from human non-engrafting hemangio cells.For example, the invention contemplates compositions and preparationscomprising one or more hematopoietic and/or endothelial cell typedifferentiated from a non-engrafting hemangio cells. Exemplaryhematopoietic cell types include hematopoietic stem cells, platelets,RBCs, lymphocytes, megakaryocytes, and the like. By way of furtherexamples, the invention contemplates compositions and preparationscomprising one or more other cell type, such as one or more partially orterminally differentiated mesodermal cell type, differentiated fromnon-engrafting hemangio cells.

In certain embodiments of any of the foregoing, the invention provides acryopreserved preparation of human non-engrafting hemangio cells orcells partially or terminally differentiated therefrom.

In certain embodiments of any of the foregoing, the invention providesfor the therapeutic use of human non-engrafting hemangio cells, orcompositions or preparations of human non-engrafting hemangio cells.Such cells and preparations can be used in the treatment of any of theconditions or diseases detailed throughout the specification, as well asin the blood banking industry. Furthermore, cells differentiated fromhuman non-engrafting hemangio cells, or compositions or preparations ofhuman non-engrafting hemangio cells, can be used therapeutically in thetreatment of any of the conditions or diseases detailed throughout thespecification.

The human non-engrafting hemangio cells of the invention can be usedtherapeutically. Additionally or alternatively, human non-engraftinghemangio cells can be used to study development of endothelial andhematopoietic lineages or in screening assays to identify factors thatcan be used, for example, to (i) maintain human non-engrafting hemangiocells or (ii) to promote differentiation of human non-engraftinghemangio cells to one or more partially or terminally differentiatedcell types. Furthermore, human non-engrafting hemangio cells can be usedto generate one or more partially or terminally differentiated celltypes for in vitro or in vivo use.

The human non-engrafting hemangio cells of the invention can be used inany of the methods or application described in the present applicationincluding, but not limited to, in the treatment of any of the diseasesor conditions described herein. Exemplary diseases and conditions arefurther discussed in U.S. application Ser. No. 11/787,262, which isincorporated by reference in its entirety. Further, humanhemangio-colony forming cells and non-engrafting hemangio cells can beused to produce differentiated hematopoietic cell types, includingfunctional red blood cells.

Cell Preparations Comprising Hemangioblasts Expanded In Vitro

In certain embodiments of the present invention, mammalian (includinghuman) non-engrafting hemangio cells are expanded to reach commercialquantities and are used in various therapeutic and clinicalapplications. In particular embodiments, non-engrafting hemangio cellsare expanded to reach cell numbers on the order of 10,000 to 4 million(or more). These cell numbers may be reached within 3-4 days of startingthe initial preparations. Accordingly, the present invention relates topreparations comprising large numbers of non-engrafting hemangio cells,said preparations comprising at least 10,000, 50,000, 100,000, 500,000,a million, 2 million, 3 million or 4 million cells.

This invention also provides for a solution, a composition, and apreparation comprising large numbers of non-engrafting hemangio cells,said solution, said composition, and said preparation comprising atleast 10,000, 50,000, 100,000, 500,000, a million, 2 million, 3 millionor 4 million cells. The non-engrafting hemangio cells could be human.The solutions can be purified, substantially purified, or mixtures withother progenitor cells types including, but not limited tohemangio-colony forming cells.

Other aspects of the present invention relate to differentiating thenon-engrafting hemangio cells obtained by the methods disclosed hereininto hematopoietic or endothelial cell lineages, or both, that aresubsequently used in clinical applications. Thus, the present inventionalso relates to cell preparations comprising large numbers of partiallyor terminally differentiated cell types.

Compositions and preparations comprising large numbers (e.g., thousandsor millions) of non-engrafting hemangio cells may be obtained byexpanding non-engrafting hemangio cells that are obtained as describedabove. Accordingly, the invention pertains to compositions andpreparations comprising large numbers of non-engrafting hemangio cellsachieved by expanding ES cells (such as human ES cells) ornon-engrafting hemangio cells obtained from cord blood, peripheral bloodor bone marrow. Further, as the methods of expansion may be applied tonon-engrafting hemangio cells of mouse, rat, bovine, or non-humanprimate origin, for example, the present invention also relates tocompositions and preparations comprising large numbers of non-engraftinghemangio cells of other species in addition to human. The non-engraftinghemangio cells to be expanded by the methods of this invention may bebi-potential, i.e., can differentiate into either endothelial cells orhematopoietic stem cells. In certain embodiments, the humannon-engrafting hemangio cells generated and expanded from human ES cellsare bi-potential. Non-engrafting hemangio cells are capable ofdifferentiating to give rise to at least hematopoietic cell types.Non-engrafting hemangio cells are, in certain embodiments, bi-potentialand capable of differentiating to give rise to at least hematopoieticcell types and endothelial cell types. As such, non-engrafting hemangiocells of the present invention are at least uni-potential, and may bebi-potential. Additionally however, non-engrafting hemangio cells mayhave a greater degree of developmental potential and can, in certainembodiments, differentiate to give rise to cell types of other lineages.In certain embodiments, the non-engrafting hemangio cells are capable ofdifferentiating to give rise to other mesodermal derivatives such ascardiac cells (for example, cardiomyocytes) and/or smooth muscle cells.

In addition, the non-engrafting hemangio cells can be used in screeningassays to identify agents that, for example, (i) promote differentiationof the cells to one or more hematopoietic cell type or (ii) promoteproliferation and/or survival of the cells to facilitate cell bankingand storage. The non-engrafting hemangio cells can also be used to studybasic developmental biology or can be compared to hemangio-colonyforming cells to ascertain the developmental differences between the tworelated stem cell populations.

Clinical and Commercial Embodiments of Human Hemangioblasts,Non-Engrafting Hemangio Cells, Hemangioblast Lineage Cells andNon-Engrafting Hemangio Lineage Cells Cell-Based Therapies

While human hemangioblast cells and non-engrafting hemangio cells havethe potential to differentiate in vivo into either hematopoietic orendothelial cells, they can be used in cell-based treatments in whicheither of these two cell types are needed or would improve treatment.Further, a patient may be treated with any therapy or treatmentcomprising hemangioblast lineage cells or and non-engrafting hemangiolineage cells (i.e., hematopoietic cells and/or endothelial cells). Thefollowing section describes methods of using the human hemangioblastsand non-engrafting hemangio cells of this invention generated andexpanded by the methods of this invention, or expanded by the methods ofthis invention.

In certain embodiments of the present invention, treatments to increaseor treat hematopoietic cells and treatments for increasing blood vesselgrowth and/or facilitating blood vessel repair are contemplated.Accordingly, in certain aspects, the present invention relates tomethods and compositions for treating a patient in need of hematopoieticcells or blood vessel growth or repair. The hemangioblasts ornon-engrafting hemangio cells may be injected into the blood vessel of asubject or be administered to the blood vessel of a subject throughoperation. The patient or the subject may be human.

In certain embodiments of the present invention, human hemangioblastcells or non-engrafting hemangio cells are used in transplantation,where HSC transplantation would otherwise be used. Such transplantationmay be used, for example, in hematopoietic reconstitution for thetreatment of patients with acute or chronic leukemia, aplastic anemiaand various immunodeficiency syndromes, as well as variousnon-hematological malignancies and auto-immune disorders, and to rescuepatients from treatment-induced aplasia following high-dose chemotherapyand/or radiotherapy. Such transplantation may be achieved in vivo or exvivo (such as in bone marrow transplant).

In other embodiments of the invention, human hemangioblast cells ornon-engrafting hemangio cells are used to treat patients in need ofhematopoietic reconstitution or hematopoietic treatment. Such patientsin include, for example, patients with thalassemias, sickle cell anemia,aplastic anemia (also called hypoplastic anemia), cytopenia, marrowhypoplasia, platelet deficiency, hematopoietic malignancies such asleukemias, paroxysmal nocturnal hemoglobinuria (PNH), and ADA (e.g.,deaminase (ADA)-deficient severe combined immunodeficiency (SCID)).

Particular embodiments of the present invention therefore relate tomethods of treating a patient in need of hematopoietic reconstitution orhematopoietic treatment using the hemangioblasts of the invention.Accordingly, the invention relates to methods of treating a patient inneed of hematopoietic reconstitution or treatment comprising selecting apatient in need thereof, generating and expanding or expanding humanhemangioblasts or non-engrafting hemangio cells according to the methodsof the present invention, and administering the human hemangioblasts orthe non-engrafting hemangio cells into the patient. Alternatively, themethod may comprise differentiating the generated and expanded orexpanded human hemangioblasts or non-engrafting hemangio cells intohuman hematopoietic cells and subsequently administering thehematopoietic cells to the patient.

Alternative embodiments include methods in which human hemangioblasts ornon-engrafting hemangio cells are produced on a large scale and storedprior to the selection of a patient in need thereof. Thus, otherembodiments of the invention relate to methods of treating a patient inneed of hematopoietic reconstitution or treatment comprising selecting apatient in need thereof, placing an order for human hemangioblasts ornon-engrafting hemangio cells already isolated and expanded according tothe methods described above, and administering said human hemangioblastsor non-engrafting hemangio cells to the patient. Likewise, the methodmay comprise differentiating said human hemangioblasts or non-engraftinghemangio cells into human hematopoietic cells and administering saidhematopoietic cells to the patient. In additional embodiments,hemangioblasts or non-engrafting hemangio cells hemizygous or homozygousfor at least one MHC allele are grown, optionally grown to commercialquantities, and optionally stored by a business entity. When a patientpresents a need for such cells, hemangioblast lineage cells ornon-engrafting hemangio lineage cells, a clinician or hospital willplace an order with the business for such cells.

Because the human hemangioblast cells and non-engrafting hemangio cellsof the invention will proliferate and differentiate into endothelialcells under an angiogenic microenvironment, the human hemangioblastcells may be used in a therapeutic manner to provide new blood vesselsor to induce repair of damaged blood vessels at a site of injury in apatient. Thus in certain aspects, the present invention relates tomethods of promoting new blood vessel growth or repairing injuredvasculature. The human hemangioblasts or non-engrafting hemangio cellsof the present invention may be used to treat endothelial injury, suchas myocardium infarction, stroke and ischemic brain, ischemic limbs andskin wounds including ischemic limbs and wounds that occur in diabeticanimals or patients, and ischemic reperfusion injury in the retina.Other ischemic conditions that may be treated with the hemangioblasts ornon-engrafting hemangio cells of the present invention include renalischemia, pulmonary ischemia, and ischemic cardiomyopathy.Hemangioblasts may also be used to help repair injured blood vesselsfollowing balloon angioplasty or deployment of an endovascular stent.Hemangioblasts or non-engrafting hemangio cells may additionally be usedin tissue grafting, surgery and following radiation injury. Further, thehemangioblasts or non-engrafting hemangio cells may be used to treatand/or prevent progression of atherosclerosis as well as to repairendothelial cell damage that occurs in systemic sclerosis and Raynaud'sphenomenon (RP) (Blann et al., 1993 J Rheumatol. (20):1325-30).

Accordingly, the invention provides various methods involved inproviding blood vessel growth or repair to a patient in need thereof. Inone embodiment, the invention provides for a method for inducingformation of new blood vessels in an ischemic tissue in a patient inneed thereof, comprising administering to said patient an effectiveamount of the purified preparation of human hemangioblast cells ornon-engrafting hemangio cells described above to induce new blood vesselformation in said ischemic tissue. Thus certain aspects of the presentinvention provide a method of enhancing blood vessel formation in apatient in need thereof, comprising selecting the patient in needthereof, isolating human hemangioblast cells or non-engrafting hemangiocells as described above, and administering the hemangioblast cells ornon-engrafting hemangio cells to the patient. In yet another aspect, thepresent invention provides a method for treating an injured blood vesselin a patient in need thereof, comprising selecting the patient in needthereof, expanding or generating and expanding human hemangioblast cellsor non-engrafting hemangio cells as described above, and administeringthe hemangioblast cells or non-engrafting hemangio cells to the patient.In addition to the aforementioned embodiments, the hemangioblasts ornon-engrafting hemangio cells may be produced on a large scale andstored prior to the selection of patient in need of hemangioblasts. Infurther embodiments, hemangioblasts hemizygous or homozygous for atleast one MHC allele are grown, optionally grown to commercialquantities, and optionally stored before a patient is selected forhemangioblast or non-engrafting hemangio cell treatment. Any of theaforementioned hemangioblasts, non-engrafting hemangio cells, or cellpreparations of these cells may be administered directly into thecirculation (intravenously). In certain embodiments (e.g., wherevascular repair is necessary in the eye, such as in the treatment ofischemia/reperfusion injury to the retina), the hemangioblast cells,non-engrafting hemangio cells, or cell preparations of these cells maybe administered by intra-vitreous injection.

Administration of the solutions or preparations of hemangioblasts,non-engrafting hemangio cells, and derivative cells thereof may beaccomplished by any route and may be determined on a case by case basis.Also, an effective amount to be administered of these solutions orpreparations of hemangioblasts or derivative cells thereof is an amountthat is therapeutically effective and may be determined on a case bycase basis.

In further aspects, hemangioblast lineage cells or non-engraftinghemangio lineage cells are used in therapeutic applications, includingin the treatment of the indications described above, for example.Accordingly, hemangioblasts or non-engrafting hemangio cells generatedand expanded or expanded by the methods described herein aredifferentiated in vitro first to obtain hematopoietic and/or endothelialcells, and then to obtain cells that are further differentiated in thesetwo lineages. These cells may be subsequently administered to a subjector patient to treat hematopoietic conditions or for hematopoieticreconstitution, or for the treatment of ischemia or vascular injury, forexample.

HSCs derived from the human hemangioblasts or non-engrafting hemangiocells obtained by the methods disclosed herein are grown further toexpand the HSCs and/or to derive other hematopoietic lineage cell types.Certain aspects of the present invention relate to the use of HSCsderived from the hemangioblasts or non-engrafting hemangio cells intransplantation. In additional embodiments, differentiated hematopoieticcells (such as, for example, granulocytes, erythrocytes, myeloid cells,megakaryocytes, platelets, macrophages, mast cells and neutrophils(Wiles and Keller 1991 Development (111): 259)) are used in varioustreatments such as transfusion therapy or for the treatment ofinfections. Accordingly, other embodiments of the present inventionrelate to methods of treating a patient in need of hematopoieticreconstitution or treatment using the HSCs or hematopoietic lineagecells derived from hemangioblasts of the invention.

In certain aspects, therefore, the present invention relates to methodsof treating a patient in need of hematopoietic cells or treatmentcomprising selecting a patient in need thereof, expanding or isolatingand expanding human hemangioblasts or non-engrafting hemangio cellsaccording to the methods of the present invention, differentiating saidhemangioblast cells or non-engrafting hemangio cells into hematopoieticstem cells and/or mature hematopoietic cells, and administering thehematopoietic cells to the patient.

In other aspects of the invention, the hemangioblasts or non-engraftinghemangio cells are grown to give rise to endothelial cells according tothe methods disclosed herein. The endothelial may subsequently be usedto provide new blood vessels or to induce repair of damaged bloodvessels at a site of injury in a patient. Thus in certain aspects, thepresent invention relates to methods of promoting new blood vesselgrowth or repairing injured vasculature in which endothelial cellsderived from hemangioblasts or non-engrafting hemangio cells are used asa therapy. The endothelial cells may be used to treat endothelialinjury, such as myocardium infarction and pulmonary ischemia, stroke andischemic brain, ischemic limbs and skin wounds including ischemic limbsand wounds that occur in diabetic animals or patients, ischemicreperfusion injury in the retina, renal ischemia. The endothelial cellsmay also be used to help repair injured blood vessels following balloonangioplasty or deployment of an endovascular stent as well as ingrafting, surgery and following radiation injury. Further, theendothelial cells may be used to treat and/or prevent progression ofatherosclerosis as well as to repair endothelial cell damage that occursin systemic sclerosis and Raynaud's phenomenon.

The endothelial cell may be further differentiated and those cells, asappropriate, may be used in treating one or more of the “endothelialcell” disease or conditions, such as those listed in the precedingparagraph.

Accordingly, certain aspects of the invention relate to methods oftreating a patient with endothelial or vascular injury or in need ofblood vessel growth or repair comprising selecting a patient in needthereof, expanding or isolating and expanding human hemangioblasts ornon-engrafting hemangio cells according to the methods of the presentinvention, differentiating said hemangioblast cells or non-engraftinghemangio cells into endothelial cells, and administering the endothelialcells to the patient.

Blood Banking

Another aspect of the present invention provides methods of producinghematopoietic cells suitable for transfusion. Although such cells andmethods have numerous uses, a particularly important use would be inimproving the availability of blood for transfusions. In certainpreferred embodiments, the invention provides red blood cellsdifferentiated from hemangioblasts/hemangio-colony forming units ornon-engrafting hemangio cells. Such differentiated red blood cells couldbe used for transfusions.

Further aspects of the invention relate to methods of generatingdifferentiated hematopoietic cells from hemangioblasts/hemangio-colonyforming units or non-engrafting hemangio cells for use in bloodtransfusions for those in need thereof. In certain embodiments,differentiated hematopoietic cells are transfused to treat trauma, bloodloss during surgery, blood diseases such as anemia, Sickle cell anemia,or hemolytic diseases, or malignant disease. In certain embodiments, redblood cells are transfused to treat trauma, blood loss during surgery,or blood diseases such as anemia, Sickle cell anemia, or hemolyticdisease. In certain embodiments, platelets are transfused to treatcongenital platelet disorders or malignant disease. In certainembodiments, a mixed population of red blood cells and platelets aretransfused.

It should be noted that many differentiated hematopoietic cell types,particularly red blood cells, typically exist in vivo as a mixedpopulation. Specifically, circulating red blood cells of varying levelsof age and differentiation are found in vivo. Additionally, red bloodcells mature over time so as to express less fetal hemoglobulin and moreadult hemoglobin. The present invention contemplates transfusion ofeither purified populations of red blood cells or of a mixed populationof red blood cells having varying levels of age and levels ofdifferentiation. In particular embodiments, the invention contemplatestransfusion of red blood cells expressing fetal hemoglobin (hemoglobinF).

This invention provides a method for producing differentiatedhematopoietic cells from human hemangio-colony forming cells andnon-engrafting hemangio cells in vitro, said method comprising the stepsof:

(a) providing human hemangio-colony forming cells or non-engraftinghemangio cells; and

b) differentiating said hemangio-colony forming cells or non-engraftinghemangio cells into differentiated hematopoietic cells.

This invention also provides a method for performing blood transfusionsusing hematopoietic cells that were differentiated in vitro from humanhemangio-colony forming cells or non-engrafting hemangio cells, saidmethod comprising the steps of:

(a) providing human hemangio-colony forming cells or non-engraftinghemangio cells;

(b) differentiating said hemangio-colony forming cells or non-engraftinghemangio cells into differentiated hematopoietic cells; and

(c) performing blood transfusions with said differentiated hematopoieticcells.

This invention also provides a method for performing blood transfusionsusing hematopoietic cells that had been differentiated in vitro fromhuman hemangio-colony forming cells, said method comprising the stepsof:

(a) culturing a cell culture comprising human embryonic stem cells inserum-free media in the presence of at least one growth factor in anamount sufficient to induce the differentiation of said embryonic stemcells into embryoid bodies;

(b) adding at least one growth factor to said culture comprisingembryoid bodies and continuing to culture said culture in serum-freemedia, wherein said growth factor is in an amount sufficient to expandhuman hemangio-colony forming cells or non-engrafting hemangio cells insaid embryoid bodies culture;

(c) differentiating said hemangio-colony forming cells or non-engraftinghemangio cells into differentiated hematopoietic cells; and

(d) performing blood transfusions with said differentiated hematopoieticcells.

In certain embodiments, said stem cells, embryoid bodies andhemangio-colony forming are grown in serum-free media throughout steps(a) and (b) of said method.

This invention also provides a method for performing blood transfusionsusing hematopoietic cells that had been differentiated in vitro fromhuman hemangio-colony forming cells, said method comprising the stepsof:

(a) culturing a cell culture comprising human pluripotent stem cells inserum-free media in the presence of at least one growth factor in anamount sufficient to induce the differentiation of said pluripotent stemcells into embryoid bodies;

(b) adding at least one growth factor to said culture comprisingembryoid bodies and continuing to culture said culture in serum-freemedia, wherein said growth factor is in an amount sufficient to expandhuman hemangio-colony forming cells or non-engrafting hemangio cells insaid embryoid bodies culture;

(c) disaggregating said embryoid bodies into single cells;

(d) adding at least one growth factor to said culture comprising saidsingle cells and continuing to culture said culture in serum-free media,wherein said growth factor is in an amount sufficient to expand humanhemangio-colony forming cells or non-engrafting hemangio cells in saidculture comprising said single cells;

(e) differentiating said hemangio-colony forming cells or non-engraftinghemangio cells into differentiated hematopoietic cells; and

(f) performing blood transfusions with said differentiated hematopoieticcells.

In certain embodiments, said pluripotent stem cells, embryoid bodies,hemangio-colony forming cells, non-engrafting hemangio cells and singlecells are grown in serum-free media throughout steps (a)-(d) of saidmethod.

In certain embodiments, the pluripotent stem cell is an embryonic stemcell.

In certain embodiments, the growth factor is a protein that comprises ahomeobox protein, or a functional variant or an active fragment thereof.In certain embodiments, the homeobox protein comprises a HOXB4 protein,or a functional variant or an active fragment thereof.

In certain embodiments, the differentiated hematopoietic cells areproduced as a single cell type such as red blood cells, platelets, andphagocytes. Note, however, that when a single cell type is produced, thecell type may be heterogeneous in terms of the level of maturity ordifferentiation of the particular cell type. By way of example,differentiated red blood cells may be heterogeneous in terms of level ofmaturity and cellular age. Without being bound by theory, suchheterogeneity of erythrocytic cells may be beneficial because it mimicsthe way in which red blood cells are found in vivo.

In certain embodiments, the single cell types are mixed to equal theproportion of differentiated cell types that is found in blood. Incertain embodiments, multiple differentiated hematopoietic cell typesare produced in the same step. In certain embodiments, the phagocyte isselected from: granulocytes, neutrophils, basophils, eosinophils,lymphocytes or monocytes. In certain embodiments, the hematopoietic celltypes are produced in a proportion approximately equal to the proportionof differentiated hematopoietic cell types found in blood, 96% red bloodcells, 1% platelets, and 3% phagocytes. In certain embodiments, plasmais added to the differentiated hematopoietic cells before transfusion.In certain embodiments, packed cells, for example packed red bloodcells, are transfused in the absence or substantial absence of plasma.

In certain embodiments, the differentiated hematopoietic cells producedfrom the methods of the application are functional. In certainembodiments, the platelets produced from the methods of the applicationare functional. In certain embodiments, the phagocytes produced from themethods of the application are functional. In certain embodiments, thered blood cells produced from the methods of the application arefunctional. In certain embodiments, the red blood cells expresshemoglobin F prior to transfusion. In certain embodiments, the red bloodcells carry oxygen. In certain embodiments, the red blood cells have alifespan equal to naturally derived red blood cells. In certainembodiments, the red blood cells have a lifespan that is 75% of that ofnaturally derived red blood cells. In certain embodiments, the red bloodcells have a lifespan that is 50% of that of naturally derived red bloodcells. In certain embodiments, the red blood cells have a lifespan thatis 25% of that of naturally derived red blood cells.

In certain embodiments, the methods of the application produce 1×10⁶cells per 100 mm dish. In certain embodiments, 2×10⁶ cells are producedper 100 mm dish. In certain embodiments, 3×10⁶ cells are produced per100 mm dish. In certain embodiments, 4×10⁶ cells are produced per 100 mmdish. In certain embodiments, 5×10⁶ cells are produced per 100 mm dish.In certain embodiments, 6×10⁶ cells are produced per 100 mm dish. Incertain embodiments, 7×10⁶ cells are produced per 100 mm dish. Incertain embodiments, 8×10⁶ cells are produced per 100 mm dish. Incertain embodiments, 9×10⁶ cells are produced per 100 mm dish. Incertain embodiments, 1×10⁷ cells are produced per 100 mm dish. Incertain embodiments, 5×10⁷ cells are produced per 100 mm dish. Incertain embodiments, 1×10⁸ cells are produced per 100 mm dish.

In certain embodiments, the differentiation step is performed usingconditions known to one of skill in the art as discussed above. Incertain embodiments, the differentiation step is performed using methodsspecific to differentiate cells into red blood cells (see WO20051118780,herein incorporated by reference). In certain embodiments, thedifferentiation step is performed using methods specific todifferentiate cells into platelets. In certain embodiments, thedifferentiation step is performed using methods specific todifferentiate cells into leukocytes.

Differentiation agents which can be used according to the presentinvention include cytokines such as interferon-alpha A, interferon-alphaA/D, interferon-.beta., interferon-gamma, interferon-gamma-inducibleprotein-10, interleukin-1, interleukin-2, interleukin-3, interleukin-4,interleukin-5, interleukin-6, interleukin-7, interleukin-8,interleukin-9, interleukin-10, interleukin-1, interleukin-12,interleukin-13, interleukin-15, interleukin-17, keratinocyte growthfactor, leptin, leukemia inhibitory factor, macrophagecolony-stimulating factor, and macrophage inflammatory protein-1 alpha.

Differentiation agents according to the invention also include growthfactors such as 6Ckine (recombinant), activin A, AlphaA-interferon,alpha-interferon, amphiregulin, angiogenin, B-endothelial cell growthfactor, beta cellulin, B-interferon, brain derived neurotrophic factor,Cl0 (recombinant), cardiotrophin-1, ciliary neurotrophic factor,cytokine-induced neutrophil chemoattractant-1, endothelial cell growthsupplement, eotaxin, epidermal growth factor, epithelial neutrophilactivating peptide-78, erythropoietin, estrogen receptor-alpha, estrogenreceptor-B, fibroblast growth factor (acidic/basic, heparin stabilized,recombinant), FLT-3/FLK-2 ligand (FLT-3 ligand), gamma-interferon, glialcell line-derived neurotrophic factor, Gly-His-Lys, granulocytecolony-stimulating factor, granulocyte macrophage colony-stimulatingfactor, GRO-alpha/MGSA, GRO-B, GRO-gamma, HCC-1, heparin-bindingepidermal growth factor like growth factor, hepatocyte growth factor,heregulin-alpha (EGF domain), insulin growth factor binding protein-1,insulin-like growth factor binding protein-1/IGF-1 complex, insulin-likegrowth factor, insulin-like growth factor II, 2.5S nerve growth factor(NGF), 7S-NGF, macrophage inflammatory protein-1B, macrophageinflammatory protein-2, macrophage inflammatory protein-3 alpha,macrophage inflammatory protein-3B, monocyte chemotactic protein-1,monocyte chemotactic protein-2, monocyte chemotactic protein-3,neurotrophin-3, neurotrophin-4, NGF-B (human or rat recombinant),oncostatin M (human or mouse recombinant), pituitary extract, placentagrowth factor, platelet-derived endothelial cell growth factor,platelet-derived growth factor, pleiotrophin, rantes, stem cell factor,stromal cell-derived factor 1B/pre-B cell growth stimulating factor,thrombopoetin, transforming growth factor alpha, transforming growthfactor-B1, transforming growth factor-B2, transforming growth factor-B3,transforming growth-factor-B5, tumor necrosis factor (alpha and B), andvascular endothelial growth factor.

Differentiation agents according to the invention also include hormonesand hormone antagonists, such as 17B-estradiol, adrenocorticotropichormone, adrenomedullin, alpha-melanocyte stimulating hormone, chorionicgonadotropin, corticosteroid-binding globulin, corticosterone,dexamethasone, estriol, follicle stimulating hormone, gastrin 1,glucagon, gonadotropin, hydrocortisone, insulin, insulin-like growthfactor binding protein, L-3,3′,5′-triiodothyronine,L-3,3′,5-triiodothyronine, leptin, leutinizing hormone, L-thyroxine,melatonin, MZ-4, oxytocin, parathyroid hormone, PEC-60, pituitary growthhormone, progesterone, prolactin, secretin, sex hormone bindingglobulin, thyroid stimulating hormone, thyrotropin releasing factor,thyroxine-binding globulin, and vasopressin.

In addition, differentiation agents according to the invention includeextracellular matrix components such as fibronectin, proteolyticfragments of fibronectin, laminin, thrombospondin, aggrecan, andsyndezan.

Differentiation agents according to the invention also includeantibodies to various factors, such as anti-low density lipoproteinreceptor antibody, anti-progesterone receptor, internal antibody,anti-alpha interferon receptor chain 2 antibody, anti-c-c chemokinereceptor 1 antibody, anti-CD 118 antibody, anti-CD 119 antibody,anti-colony stimulating factor-1 antibody, anti-CSF-1 receptor/c-finsantibody, anti-epidermal growth factor (AB-3) antibody, anti-epidermalgrowth factor receptor antibody, anti-epidermal growth factor receptor,phospho-specific antibody, anti-epidermal growth factor (AB-1) antibody,anti-erythropoietin receptor antibody, anti-estrogen receptor antibody,anti-estrogen receptor, C-terminal antibody, anti-estrogen receptor-Bantibody, anti-fibroblast growth factor receptor antibody,anti-fibroblast growth factor, basic antibody, anti-gamma-interferonreceptor chain antibody, anti-gamma-interferon human recombinantantibody, anti-GFR alpha-1 C-terminal antibody, anti-GFR alpha-2C-terminal antibody, anti-granulocyte colony-stimulating factor (AB-1)antibody, anti-granulocyte colony-stimulating factor receptor antibody,anti-insulin receptor antibody, anti-insulin-like growth factor-1receptor antibody, anti-interleukin-6 human recombinant antibody,anti-interleukin-1 human recombinant antibody, anti-interleukin-2 humanrecombinant antibody, anti-leptin mouse recombinant antibody, anti-nervegrowth factor receptor antibody, anti-p60, chicken antibody,anti-parathyroid hormone-like protein antibody, anti-platelet-derivedgrowth factor receptor antibody, anti-platelet-derived growth factorreceptor-B antibody, anti-platelet-derived growth factor-alpha antibody,anti-progesterone receptor antibody, anti-retinoic acid receptor-alphaantibody, anti-thyroid hormone nuclear receptor antibody, anti-thyroidhormone nuclear receptor-alpha 1/Bi antibody, anti-transferrinreceptor/CD71 antibody, anti-transforming growth factor-alpha antibody,anti-transforming growth factor-B3 antibody, anti-tumor necrosisfactor-alpha antibody, and anti-vascular endothelial growth factorantibody.

This invention also provides a library of differentiated hematopoieticcells that can provide matched cells to potential patient recipients asdescribed above. In certain embodiments, the cells are stored frozen.Accordingly, in one embodiment, the invention provides a method ofconducting a pharmaceutical business, comprising the step of providingdifferentiated hematopoietic cell preparations that are homozygous forat least one histocompatibility antigen, wherein cells are chosen from abank of such cells comprising a library of human hemangio-colony formingcells or non-engrafting hemangio cells that can be expanded by themethods disclosed herein, wherein each hemangio-colony forming cell ornon-engrafting hemangio cells preparation is hemizygous or homozygousfor at least one MHC allele present in the human population, and whereinsaid bank of hemangio-colony forming cells or non-engrafting hemangiocells comprises cells that are each hemizygous or homozygous for adifferent set of MHC alleles relative to the other members in the bankof cells. As mentioned above, gene targeting or loss of heterozygositymay be used to generate the hemizygous or homozygous MHC allele stemcells used to derive the hemangio-colony forming cells or non-engraftinghemangio cells. In certain embodiments, hemangio-colony forming cells ornon-engrafting hemangio cells of all blood types are included in thebank. In certain embodiments, hemangio-colony forming cells ornon-engrafting hemangio cells are matched to a patient to ensure thatdifferentiated hematopoietic cells of the patient's own blood type areproduced. In certain embodiments, hemangio-colony forming cells ornon-engrafting hemangio cells are negative for antigenic factors A, B,Rh, or any combination thereof. In certain embodiments, thedifferentiated hematopoietic cells are universal donor cells. By way ofexample, hematopoietic cells that are type O and Rh negative can beuniversally used for blood transfusion. In certain embodiments, theinvention provides methods for producing type O, Rh negative red bloodcells for universal transfusion.

In certain embodiments, red blood cells differentiated fromhemangio-colony forming cells or non-engrafting hemangio cells expressfetal hemoglobin. Transfusion of red blood cells that express fetalhemoglobin may be especially useful in the treatment of Sickle cellanemia. As such, the present invention provides improved methods fortreating Sickle cell anemia.

In one embodiment, after a particular hemangio-colony forming cellpreparation or a non-engrafting hemangio cell preparation is chosen tobe suitable for a patient, it is thereafter expanded to reachappropriate quantities for patient treatment and differentiated toobtain differentiated hematopoietic cells prior to administering cellsto the recipient. Methods of conducting a pharmaceutical business mayalso comprise establishing a distribution system for distributing thepreparation for sale or may include establishing a sales group formarketing the pharmaceutical preparation.

In any of the foregoing, hemangio-colony forming cells or non-engraftinghemangio cells can be directly differentiated or hemangio-colony formingcells or non-engrafting hemangio cells can be frozen for later use. Incertain embodiments, the invention provides a frozen culture ofhemangio-colony forming cells or non-engrafting hemangio cells suitablefor later thawing and expansion, and also suitable for differentiationto hematopoietic or endothelial lineages.

Human hemangio-colony forming cells or non-engrafting hemangio cells canbe used to generate substantial numbers of hematopoietic cell types thatcan be used in blood transfusions. For examples, substantial numbers ofhomogeneous or heterogeneous populations RBCs and/or platelets can begenerated from human hemangio-colony forming cells. Hemangio-colonyforming cells, non-engrafting hemangio cells and hematopoietic celltypes differentiated therefrom can be banked, as is currently done withdonated blood products, and used in transfusions and other treatments.Banking of these products will help alleviate the critical shortage ofdonated blood products. Additionally, hemangio-colony forming cells,non-engrafting hemangio cells and derivative products can be geneticallymanipulated in vitro to provide universal donor blood products.

As such, in certain aspects the invention provides a method ofconducting a blood banking business. The subject banking businessinvolves the derivation and storage (long or short term) ofhemangio-colony forming cells, non-engrafting hemangio cells and/orhematopoietic cell types (e.g., RBCs, platelets, lymphocytes, etc.)generated therefrom. Cells can be cryopreserved for long term storage,or maintained in culture for relatively short term storage. Cells can betyped and cross-matched in much the same way the currently availableblood products are typed, and the cells can be stored based on type.Additionally and in certain embodiments, cells can be modified tospecifically generate cells that are A negative and/or B negative and/orRh negative to produce cells that are universally or nearly universallysuitable for transfusion into any patient.

Note that hemangio-colony forming cells, non-engrafting hemangio cellsand/or differentiated hematopoietic cell types can be generated usingany of the methods of the invention detailed through the specification.

In certain embodiments of a method of conducting a blood bankingbusiness, the cells (hemangio-colony forming cells, non-engraftinghemangio cells and/or differentiated hematopoietic cell types) aregenerated and stored at one or more central facilities. Cells can thenbe transferred to, for example, hospitals or treatment facilities foruse in patient care. In certain other embodiments, cells are maintainedin a cryopreserved state and specifically thawed and prepared fortransfusion based on orders from hospitals or other treatmentfacilities. Such orders may be a standing order (e.g., generate andprovide a certain quantity of cells of a certain number of units

In certain embodiments, the method includes a system for billinghospitals or insurance companies for the costs associated with thebanked products.

In certain embodiments of any of the foregoing, the cells can beallocated based on cell number, volume, or any unit that permits theuser to quantify the dose being administered to patients and/or tocompare these doses to that administered during a standard bloodtransfusion.

In certain embodiments, the cells are generated, stored, andadministered as a mixed population of cells. For example, thepreparation may include cells of varying developmental stages, as wellas distinct cell types. In other embodiments, the cells are generated,stored, and/or administered as a substantially purified preparation of asingle cell type.

In certain embodiments, the preparations of cells are screened for oneor more infectious diseases. Screening may occur prior to or subsequentto generation or storage. For example, the preparations of cells may bescreened to identify hepatitis, HIV, or other blood-borne infectiousdisease that could be transmitted to recipients of these products.

Induction of Tolerance in Graft Recipients

The human hemangioblast cells generated and expanded by the methods ofthis invention, or expanded by the methods of this invention, may beused to induce immunological tolerance. Immunological tolerance refersto the inhibition of a graft recipient's immune response which wouldotherwise occur, e.g., in response to the introduction of a nonself MHCantigen (e.g., an antigen shared with the graft and the tolerizinghemangioblasts) into the recipient. Thus, tolerance refers to inhibitionof the immune response induced by a specific donor antigen as opposed tothe broad spectrum immune inhibition that may be elicited usingimmunosuppressants. Tolerance may involve humoral, cellular, or bothhumoral and cellular responses. Tolerance may include the eliminationand/or inactivation of preexisting mature donor-reactive T cells as wellas long-term (e.g. lifelong) elimination and/or inactivation of newlydeveloping donor-reactive T cells.

The methods described in the present invention of generating andexpanding human hemangioblasts offer several advantages for inducingtolerance. The methods of the present invention result in the generationof large, previously unobtainable numbers of human hemangioblasts. Largenumbers of human hemangioblasts allow induction of tolerance in graftrecipients with less toxic preconditioning protocols. Furthermore, themethods of the present invention provide for the generation of a libraryof human hemangioblasts, each of which is hemizygous or homozygous forat least one MHC allele present in the human population, wherein eachmember of said library of hemangioblast cells is hemizygous orhomozygous for a different set of MHC alleles relative to the othermembers in the library. Such a library of human hemangioblasts can beused in the selection of tolerizing human hemangioblast cells such thatcells can be selected to match any available donor graft.

Bone marrow transplantation and subsequent establishment ofhematopoietic or mixed chimerism have previously been shown to inducespecific tolerance to new tissue types derived from hematopoietic stemcells in both murine and human models. Hematopoietic or mixed chimerismrefers to the production in a recipient of hematopoietic cells derivedfrom both donor and recipient stem cells. Hence, if a recipient achieveshematopoietic chimerism, the recipient will be tolerant todonor-specific antigens. In many protocols for inducing tolerance, thetolerizing donor cells that are administered to the recipient engraftinto the bone marrow of the recipient. To create hematopoietic space inthe recipient bone marrow for the donor cells, some protocols require astep of creating hematopoietic space (e.g., by whole body irradiation),and such a step is typically toxic or harmful to the recipient. However,if very large numbers of donor tolerizing cells are available, there isevidence from rodent models that irradiation can be completelyeliminated, thereby achieving hematopoietic or mixed chimerism with theadvantage of less toxic pre-conditioning regimens. Thus, mixed chimerismcan be achieved, for example, with specific, non-myeloablative recipientconditioning.

Accordingly, as the novel methods described herein enable the productionof large numbers of human hemangioblast cells, the present inventionoffers the advantage of inducing immune tolerance with less rigorous orless toxic conditioning protocols. For example, the hematopoieticspace-creating step may be eliminated if a sufficient number oftolerizing donor cells are used.

Accordingly, in certain embodiments of the present invention, humanhemangioblast cells generated and expanded or expanded by the methodsdescribed herein may be used to induce immunological tolerance. Whilenot wishing to be bound by any theory on the mechanism, the humanhemangioblast cells may induce immunological tolerance by homing to therecipient's bone marrow and engrafting into the recipient's bone marrowin order to produce mixed chimerism.

In certain embodiments, donor human hemangioblast cells are administeredto a recipient patient (e.g., by intravenous injection) prior toimplanting a graft or transplanting an organ, tissue, or cells from thedonor into the recipient patient. In certain embodiments, humanhemangioblasts are administered to induce tolerance in patients in needthereof (e.g., graft or transplant recipients). Accordingly, in certainembodiments the method of inducing tolerance in a human recipientpatient comprises the steps of: (a) selecting a patient in need of atransplant or cellular therapy; (b) administering to said patient humanhemangioblast cells derived from a donor or that are matched to thedonor, wherein said hemangioblast cells are generated and expanded orexpanded according to the methods of this invention, and (c) implantinga donor organ, tissue, or cell graft into the recipient patient, whereinsaid hemangioblast cells induce tolerance to donor antigens. In certainembodiments, the patient will receive an organ, tissue, or cell therapy,wherein the organ, tissue, or cells are obtained from the donor or adonor cell source. For example, hemangioblast cells from a donor can be(1) expanded according to the methods described herein to generate alarge number of donor tolerizing cells, and (2) expanded anddifferentiated in vitro to obtain hematopoietic or endothelial cells ortissues, which can be subsequently implanted into the recipient patient.In other embodiments, the organ, tissue, or cell therapy is not derivedfrom donor hemangioblast cells but is matched to the donorhemangioblasts.

As used herein, the term “matched” relates to how similar the HLA typingis between the donor and the recipient (e.g., graft). In one embodiment,the term “matched” with respect to donor hemangioblast cells and graftrefers to a degree of match t the MHC class I and/or at the MHC class IIalleles such that rejection does not occur. In another embodiment, theterm “matched” with respect to donor hemangioblasts and graft refers toa degree of match at the MHC class I and/or at the MHC class II allelessuch that the donor graft is tolerized by its matching donorhemangioblast cells. In another embodiment, the term “matched” withrespect to donor hemangioblast and graft refers to a degree of match atthe MHC class I and/or at the MHC class II alleles such thatimmunosuppression is not required.

The methods described herein for inducing tolerance to an allogeneicantigen or allogeneic graft may be used where, as between the donor andrecipient, there is degree of mismatch at MHC loci or other loci, suchthat graft rejection results. Accordingly, for example, in certainembodiments, there may be a mismatch at least one MHC locus or at leastone other locus that mediates recognition and rejection, e.g., a minorantigen locus. In some embodiments, for example, the HLA alleles of therecipient and donor are mismatched and result in one or more mismatchedantigens. With respect to class I and class II MHC loci, the donor andrecipient may be, for example: matched at class I and mismatched atclass II; mismatched at class I and matched at class II; mismatched atclass I and mismatched at class II; matched at class I, matched at classII. In any of these combinations other loci which control recognitionand rejection, e.g., minor antigen loci, may be matched or mismatched.Mismatched at MHC class I means mismatched for one or more MHC class Iloci, e.g., mismatched at one or more of HLA-A, HLA-B, or HLA-C.Mismatched at MHC class II means mismatched at one or more MHC class IIloci, e.g., mismatched at one or more of a DPA, a DPB, a DQA, a DQB, aDRA, or a DRB. For example, the hemangioblasts and the graft may bematched at class II HLA-DRB1 and DQB1 alleles. The hemangioblasts andgraft may further be matched at two or more class I HLA-A, B, or C,alleles (in addition to having matched DRB1 and DQB1 alleles).

In other embodiments, the tolerizing donor cells are cells derived fromthe hemangioblasts generated and expanded or expanded by the methodsdescribed herein. According to this embodiment, donor humanhemangioblasts are differentiated in vitro to give rise to donorhematopoietic stem cells, and the donor hematopoietic stem cells arethen administered to the recipient patient to induce tolerance. In anyof the above methods, the donor hemangioblasts or hematopoietic stemcells derived therefrom and administered to said recipient prepare therecipient patient for the matched (with respect to the donor tolerizingcells) transplant or graft by inducing tolerance in said recipient.

In other embodiments, the method of inducing tolerance further comprisesthe step(s) of creating hematopoietic space (to promote engraftment ofhemangioblasts or hematopoietic stem cells derived therefrom). Inanother embodiment, the method of inducing tolerance further comprisesthe step(s) of temporarily inhibiting rejection of donor hemangioblastcells or hematopoietic stem cells derived therefrom by, for example,eliminating and/or inactivating preexisting donor-reactive T cells. Inorder to create hematopoietic space, the method may include irradiation(e.g., whole body, lymphoid, or selective thymic irradiation). Toprevent rejection of donor cells, the method may further comprise theadministration of drugs or antibodies (e.g., inhibitors of cellproliferation, anti-metabolites, or anti-T cell or anti-CD8 or anti-CD4antibodies), and/or other treatments that promote survival andengraftment of the donor cells and the formation of mixed chimerism(e.g., the administration of stromal cells or growth factors, cytokines,etc. to said recipient, or other agents that deplete or inactive therecipient's natural antibodies). In certain embodiments, theirradiation, antibodies, drugs, and/or other agents administered tocreate hematopoietic space and/or promote survival of donor cells in therecipient, is sufficient to inactivate thymocytes and/or T cells in therecipient. Such a step of creating hematopoietic space and/ortemporarily inhibiting rejection of donor cells may be performed, forexample, before the introduction of the donor hemangioblast cells tosaid recipient. Alternatively, the patient may receive an agent ormethod for blocking, eliminating, or inactivating T cells concurrentlywith the administration of the donor tolerizing cells.

In certain embodiments, a combination of hematopoietic space-creatingand immunosuppressive methods is used. For example, a recipient mayreceive an anti-T cell antibody in combination with low dose whole bodyirradiation and/or thymic irradiation. In one embodiment, the recipientmay receive anti-CD4 and anti-CD8 antibodies, followed by a mild,nonmyeloablative dose of whole body irradiation (e.g., a dose thateliminates a fraction of the recipient's bone marrow without renderingthe bone marrow unrecoverable) and selective thymic irradiation oralternatively, an additional dose of T cell-inactivating antibodies orcostimulatory blocking reagents (e.g., CTLA4-Ig and/or anti-CD40Lantibody). Following the irradiation, donor hemangioblast cells, orhematopoietic stem cells derived therefrom, may be administered to therecipient (e.g., by intravenous injection). In this embodiment, wholebody irradiation to promote engraftment of donor cells may be replacedby administering a large number of donor human hemangioblasts orhematopoietic stem cells derived therefrom. Obtaining such large numbersof donor human cells can be achieved according to the methods describedherein.

In another embodiment, treatments to deplete or inactivate recipient Tcells may help to prevent inhibition of engraftment or promote survivalof the administered donor tolerizing human hemangioblast cells. Inanother embodiment, the method may include clonal deletion ofdonor-reactive cells in the recipient patient. For example, a patientmay receive a mild dose of whole body irradiation, followed byadministration of donor human hemangioblasts and T cell costimulatoryblockade. Alternatively, a patient may receive T cell costimulatoryblockade and administration of large numbers of donor humanhemangioblast cells without receiving irradiation.

In another embodiment, tolerance may be achieved without myeloablativeconditioning of the recipient. In one embodiment, a recipient mayreceive donor human hemangioblasts in combination with anti-CD40L tofacilitate engraftment of donor hemangioblasts. For example, a recipientmay receive large numbers of donor hemangioblasts, along with anti-CD40Lmonoclonal antibody, followed within a few days by a dose of CTLA4-Ig.Such a protocol may delete donor-reactive T cells and block theCD40-CD40L interaction. The novel methods described herein forgenerating and expanding human hemangioblasts in vitro render such amild tolerance protocol feasible.

Following recipient conditioning and/or depletion or blocking ofdonor-reactive T cells, donor tolerizing human hemangioblasts generatedby the methods of the present invention are administered to therecipient. Donor human hemangioblasts may be derived from hemangioblastsobtained from a tissue or cell source from the donor. Alternatively,donor human hemangioblasts may be obtained from a different non-donorsource that is matched to the donor.

In certain embodiments, tolerance is induced in a recipient patient byadministering donor human hemangioblasts in multiple administrations(e.g., by two, three, four, or more administrations of the donor cells).Accordingly, tolerance may be induced by a method comprising multipleadministrations of donor tolerizing cells, wherein the multipleadministrations are given to the recipient within a timeframe of a weekor less.

In certain embodiments, the ability of the human hemangioblast cells ofthis invention to induce immunological tolerance may be evaluated usingdifferent experimental model systems. For example, the ability toestablish a human immune system in a SCID mouse has been used to studythe human immune response in an experimental model. It has beenpreviously shown that human fetal liver and thymus tissue may be used toreconstitute a functional human immune system in an immuno-incompetentmouse recipient. Similarly, the functional capacity of the humanhemangioblast cells of this invention can be assessed using a similarexperimental model system. For example, the ability of humanhemangioblasts to replace human fetal liver in establishing a functionalhuman immune system in the mouse can be evaluated using theabove-described experimental model. Further, in a mouse with afunctional human immune system (e.g., where a human fetal liver andthymus tissue is used to establish a human immune system in a SCID mouseto produce a hu-SCID mouse), human “donor” hemangioblasts (mismatchedwith respect to the fetal liver and thymic tissue used to establish thehu-SCID mouse) may be administered to the hu-SCID mouse, according toany of the methods described above, in order to achieve mixed chimerism.Tolerance to donor antigen can be subsequently tested upon implantationof an allograft matched with respect to the donor hemangioblasts intothese animals.

In certain embodiments, the present invention relates to cellcombinations. Effective cell combinations comprise two components: afirst cell type to induce immunological tolerance, and a second celltype that regenerates the needed function. Both cell types may beproduced by the methods of the present invention and obtained from thesame donor. For example, human hemangioblast cells from a donor may beused as the tolerizing donor cells. Cells from the donor (e.g.,embryonic stem cells, pluripotent stem cells or early progenitor cells,or hemangioblasts) may also be used to generate, for example,hematopoietic cells or endothelial cells (as described herein), neuralcells such as oligodendrocytes, hepatocytes, cardiomyocytes orcardiomyocyte precursors, or osteoblasts and their progenitors.Accordingly, the donor human hemangioblasts may be used to inducetolerance in a recipient such that the recipient is tolerant to cells ortissues derived from said donor hemangioblast cells or from said donorembryonic or pluripotent stem cells.

In another embodiment, the two cell components of the cell combinationsof the present invention may be obtained from different sources ordonors, wherein the two sources or donors are matched. For example,hemangioblasts may be generated from an embryonic stem cell source,whereas the graft cells or tissues may be obtained from a source that isdifferent from the embryonic stem cell source used to generate the humanhemangioblasts. In such embodiments, the two sources are matched.

For any of the therapeutic purposes described herein, humanhemangioblast or hematopoietic cells derived therefrom forimmunotolerance may be supplied in the form of a pharmaceuticalcomposition, comprising an isotonic excipient prepared undersufficiently sterile conditions for human administration.

Hemangioblasts in Gene Therapy

Other aspects of the invention relate to the use of hemangioblast cells,non-engrafting hemangio cells, or hematopoietic or endothelial cellsdifferentiated therefrom, or in turn cells further differentiated fromthese cells, in gene therapy. The preparation of mammalian hemangioblastcells or non-engrafting hemangio cells of the invention may be used todeliver a therapeutic gene to a patient that has a condition that isamenable to treatment by the gene product of the therapeutic gene. Thehemangioblasts and non-engrafting hemangio cells are particularly usefulto deliver therapeutic genes that are involved in or influenceangiogenesis (e.g. VEGF to induce formation of collaterals in ischemictissue), hematopoiesis (e.g. erythropoietin to induce red cellproduction), blood vessel function (e.g. growth factors to induceproliferation of vascular smooth muscles to repair aneurysm) or bloodcell function (e.g. clotting factors to reduce bleeding) or code forsecreted proteins e.g. growth hormone. Methods for gene therapy areknown in the art. See for example, U.S. Pat. No. 5,399,346 by Andersonet al. A biocompatible capsule for delivering genetic material isdescribed in PCT Publication WO 95/05452 by Baetge et al. Methods ofgene transfer into bone-marrow derived cells have also previously beenreported (see U.S. Pat. No. 6,410,015 by Gordon et al.). The therapeuticgene can be any gene having clinical usefulness, such as a gene encodinga gene product or protein that is involved in disease prevention ortreatment, or a gene having a cell regulatory effect that is involved indisease prevention or treatment. The gene products may substitute adefective or missing gene product, protein, or cell regulatory effect inthe patient, thereby enabling prevention or treatment of a disease orcondition in the patient.

Accordingly, the invention further provides a method of delivering atherapeutic gene to a patient having a condition amenable to genetherapy comprising, selecting the patient in need thereof, modifying thepreparation of hemangioblasts or non-engrafting hemangio cells so thatthe cells carry a therapeutic gene, and administering the modifiedpreparation to the patient. The preparation may be modified bytechniques that are generally known in the art. The modification mayinvolve inserting a DNA or RNA segment encoding a gene product into themammalian hemangioblast cells, where the gene enhances the therapeuticeffects of the hemangioblast cells or the non-engrafting hemangio cells.The genes are inserted in such a manner that the modified hemangioblastcell will produce the therapeutic gene product or have the desiredtherapeutic effect in the patient's body. In one embodiment, thehemangioblasts or non-engrafting hemangio cells are prepared from a cellsource originally acquired from the patient, such as bone marrow. Thegene may be inserted into the hemangioblast cells or non-engraftinghemangio cells using any gene transfer procedure, for example, naked DNAincorporation, direct injection of DNA, receptor-mediated DNA uptake,retroviral-mediated transfection, viral-mediated transfection, non-viraltransfection, lipid-mediated transfection, electrotransfer,electroporation, calcium phosphate-mediated transfection, microinjectionor proteoliposomes, all of which may involve the use of gene therapyvectors. Other vectors can be used besides retroviral vectors, includingthose derived from DNA viruses and other RNA viruses. As should beapparent when using an RNA virus, such virus includes RNA that encodesthe desired agent so that the hemangioblast cells that are transfectedwith such RNA virus are therefore provided with DNA encoding atherapeutic gene product. Methods for accomplishing introduction ofgenes into cells are well known in the art (see, for example, Ausubel,id.).

In accordance with another aspect of the invention, a purifiedpreparation of human hemangioblast cells or non-engrafting hemangiocells, in which the cells have been modified to carry a therapeuticgene, may be provided in containers or commercial packages that furthercomprise instructions for use of the preparation in gene therapy toprevent and/or treat a disease by delivery of the therapeutic gene.Accordingly, the invention further provides a commercial package (i.e.,a kit) comprising a preparation of mammalian hemangioblast cells ornon-engrafting hemangio cells of the invention, wherein the preparationhas been modified so that the cells of the preparation carry atherapeutic gene, and instructions for treating a patient having acondition amenable to treatment with gene therapy.

Other Commercial Applications and Methods

Certain aspects of the present invention pertain to the expansion ofhuman hemangioblasts and non-engrafting hemangio cells to reachcommercial quantities. In particular embodiments, human hemangioblastsand non-engrafting hemangio cells are produced on a large scale, storedif necessary, and supplied to hospitals, clinicians or other healthcarefacilities. Once a patient presents with an indication such as, forexample, ischemia or vascular injury, or is in need of hematopoieticreconstitution, human hemangioblasts or non-engrafting hemangio cellscan be ordered and provided in a timely manner. Accordingly, the presentinvention relates to methods of generating and expanding humanhemangioblasts and non-engrafting hemangio cells to attain cells on acommercial scale, cell preparations comprising human hemangioblasts ornon-engrafting hemangio cells derived from said methods, as well asmethods of providing (i.e., producing, optionally storing, and selling)human hemangioblasts or non-engrafting hemangio cells to hospitals andclinicians. Further, hemangioblast lineage cells or non-engraftinghemangio lineage cells may be produced in vitro and optionally storedand sold to hospitals and clinicians.

Accordingly certain aspects of the present invention relate to methodsof production, storage, and distribution of hemangioblasts ornon-engrafting hemangio cells expanded by the methods disclosed herein.Following human hemangioblast or non-engrafting hemangio cellsgeneration and expansion in vitro, human hemangioblasts ornon-engrafting hemangio cells may be harvested, purified and optionallystored prior to a patient's treatment. Alternatively, in situations inwhich hemangioblast or non-engrafting hemangio lineage cells aredesired, human hemangioblasts or non-engrafting hemangio cells may bedifferentiated further in vitro prior to a patient's treatment. Thus, inparticular embodiments, the present invention provides methods ofsupplying hemangioblasts or non-engrafting hemangio cells to hospitals,healthcare centers, and clinicians, whereby hemangioblasts,non-engrafting hemangio cells, hemangioblast lineage cells, ornon-engrafting hemangio lineage cells produced by the methods disclosedherein are stored, ordered on demand by a hospital, healthcare center,or clinician, and administered to a patient in need of hemangioblast,non-engrafting hemangio cells, hemangioblast lineage, or non-engraftinghemangio lineage therapy. In alternative embodiments, a hospital,healthcare center, or clinician orders human hemangioblasts ornon-engrafting hemangio cells based on patient specific data, humanhemangioblasts or non-engrafting hemangio cells are produced accordingto the patient's specifications and subsequently supplied to thehospital or clinician placing the order.

Further aspects of the invention relate to a library of hemangioblasts,non-engrafting hemangio cells, hemangioblast lineage cells, and/ornon-engrafting hemangio lineage cells that can provide matched cells topotential patient recipients. Accordingly, in one embodiment, theinvention provides a method of conducting a pharmaceutical business,comprising the step of providing hemangioblast or non-engraftinghemangio cell preparations that are homozygous for at least onehistocompatibility antigen, wherein cells are chosen from a bank of suchcells comprising a library of human hemangioblasts or non-engraftinghemangio cells that can be expanded by the methods disclosed herein,wherein each hemangioblast or non-engrafting hemangio cell preparationis hemizygous or homozygous for at least one MHC allele present in thehuman population, and wherein said bank of hemangioblast cells ornon-engrafting hemangio cells comprises cells that are each hemizygousor homozygous for a different set of MHC alleles relative to the othermembers in the bank of cells. As mentioned above, gene targeting or lossof heterozygosity may be used to generate the hemizygous or homozygousMHC allele stem cells used to derive the hemangioblasts. In oneembodiment, after a particular hemangioblast or non-engrafting hemangiocell preparation is chosen to be suitable for a patient, it isthereafter expanded to reach appropriate quantities for patienttreatment. Such methods may further comprise the step of differentiatingthe hemangioblasts or non-engrafting hemangio cells to obtainhematopoietic and/or endothelial cells prior to administering cells tothe recipient. Methods of conducting a pharmaceutical business may alsocomprise establishing a distribution system for distributing thepreparation for sale or may include establishing a sales group formarketing the pharmaceutical preparation.

Other aspects of the invention relate to the use of the humanhemangioblasts and non-engrafting hemangio cells of the presentinvention as a research tool in settings such as a pharmaceutical,chemical, or biotechnology company, a hospital, or an academic orresearch institution. For example, human hemangioblasts, non-engraftinghemangio cells and derivative cells thereof (e.g., endothelial cells)may be used to screen and evaluate angiogenic and anti-angiogenicfactors or may be used in tissue engineering. In addition, because thehemangioblasts and non-engrafting hemangio cells obtained and expandedby the methods disclosed herein have dual potential to differentiateinto hematopoietic and endothelial cells, they may be used for thecellular and molecular biology of hematopoiesis and vasculogenesis.Further, the human hemangioblasts and non-engrafting hemangio cells maybe used for the discovery of novel markers of these cells, genes, growthfactors, and differentiation factors that play a role in hematopoiesisand vasculogenesis, or for drug discovery and the development ofscreening assays for potentially toxic or protective agents.

In other embodiments of the present invention, hemangioblast andnon-engrafting hemangio lineage cells (such as blood cells) are alsoused commercially. Hematopoietic cells may be used to generate bloodproducts, such as hemoglobin and growth factors, that may be used forclinical and research applications.

The present invention also includes methods of obtaining human ES cellsfrom a patient and then generating and expanding human hemangioblasts ornon-engrafting hemangio cells derived from the ES cells. Thesehemangioblasts and non-engrafting hemangio cells may be stored. Inaddition, these hemangioblasts and non-engrafting hemangio cells may beused to treat the patient from which the ES were obtained or a relativeof that patient.

As the methods and applications described above relate to treatments,pharmaceutical preparations, and the storing of hemangioblasts ornon-engrafting hemangio cells, the present invention also relates tosolutions of hemangioblasts and non-engrafting hemangio cells that aresuitable for such applications. The present invention accordinglyrelates to solutions of hemangioblasts and non-engrafting hemangio cellsthat are suitable for injection into a patient. Such solutions maycomprise cells formulated in a physiologically acceptable liquid (e.g.,normal saline, buffered saline, or a balanced salt solution). A solutionmay optionally comprise factors that facilitate cell differentiation invivo. A solution may be administered to a patient by vascularadministration (e.g., intravenous infusion), in accordance with artaccepted methods utilized for bone marrow transplantation. In someembodiments, the cell solution is administered into a peripheral vein, asuperficial peripheral vein, or alternatively, by central venousadministration (e.g., through a central venous catheter). The number ofcells in the solution may be at least about 10² and less than about 10⁹cells. In other embodiments, the number of cells in the solution mayrange from about 10¹, 10², 5×10², 10³, 5×10³, 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸to about 5×10², 10³, 5×10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹, where theupper and lower limits are selected independently, except that the lowerlimit is always less than the upper limit. Further, the cells may beadministered in a single or in multiple administrations.

The present invention will now be more fully described with reference tothe following examples, which are illustrative only and should not beconsidered as limiting the invention described above.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 Materials and Methods

Generation and Expansion of Erythroid Cells from hESCs ViaHemangioblasts

Four human ESC lines were used in the current study:, H1 (NationalInstitutes of Health registered as WA01), MA01 and MA99 (derived atAdvanced Cell Technology), and HuES-3 (established by Cowan et al. (N.Engl. J. Med. 2004; 350:1353-1356) and obtained from the Harvard StemCell Institute). hESCs were grown on mitomycin C-treated mouse embryonicfibroblast (MEF) in complete hESC media until they reached 80%confluence. A four step procedure was used for the generation andexpansion of erythroid cells from hESCs.

Step 1, EB formation and hemangioblast precursor induction (Day [−]3.5-0): To induce hemangioblast precursor (mesoderm) formation, EBs wereformed by plating one well of hESCs per EB culture well (ultra-lowsix-well plates, Corning) in 3-4 ml serum free Stemline media (Sigma)with BMP-4, VEGF165 (50 ng/ml each, R&D Systems) and basic FGF (20ng/ml, Invitrogen). Half of the media was refreshed 48 hours later withthe addition of SCF, Tpo and FLT3 ligand (20 ng/ml each R&D Systems).

Step 2, Hemangioblast expansion (Day 0-10): After 3.5 days, EBs werecollected and dissociated with trypsin. A single cell suspension wasobtained by passing the cells through a G21 needle three times andfiltering through a 40 μm filter. After resuspending in Stemline IImedium, the cells were mixed with blast-colony growth media (BGM) (5×10⁵cells/ml) and plated in 100 mm ultra low dishes (10 ml/dish). Thecultures were expanded for 9-10 days in BGM. The addition of 20 ng/ml ofbFGF and 2 ug/ml of the recombinant tPTD-HOXB4 fusion protein to BGM wasfound to significantly enhance hematopoietic cell proliferation. HOXB4protein has been shown to promote hematopoietic development in bothmouse and human ESC differentiation systems (Helgason et al., Blood1996; 87:2740-2749; Kyba et al., Cell 2002; 109:29-37; Wang et al.,Proc. Natl. Acad. Sci. U.S.A 2005; 102:19081-19086; Bowles et al., StemCells 2006; 24:1359-1369; Pilat et al., Proc. Natl. Acad. Sci. U.S.A2005; 102:12101-12106; Lu et al., Stem Cells Dev. 2007; 16:547-560). Thegrape-like blast colonies were usually visible by microscopy after 4-6days, and expanded rapidly outward. Additional BGM was added to keep thedensity of blast cells at 1-2×10⁶ cells/ml.

Step 3, Erythroid cell differentiation and expansion (Day 11-20): At theend of step 2, the cell density was often very high (≧2×10⁶/ml). Equalvolumes of BGM, containing 3 units/ml of Epo (total Epo is 6 units/ml)without HOXB4, were added to supplement the existing BGM. The blastcells were further expanded and differentiated into erythroid cells foran additional 5 days. For further expansion, the erythroid cells weretransferred into 150 mm Petri dishes and Stemline 1′-based mediumcontaining SCF (100 ng/ml), Epo (3 unit/ml) and 0.5% methylcelluloseadded every 2-3 days. (When the cells reached confluence, it was veryimportant to split the cells at a ratio of 1:3 to allow maximumexpansion for an additional 7 days [cell density 2-4×10⁶/ml]).

Step 4, Enrichment of erythroid cells (Day 21): Erythroid cells obtainedfrom step 3 were diluted in 5 volumes of IMDM plus 0.5% BSA medium andcollected by centrifugation at 1000 rpm for 5 minutes. The cell pelletswere washed twice with IMDM medium containing 0.5% BSA, and plated intissue culture flasks overnight to allow nonerythroid cells (usually thelarger cells) to attach. The non-adherent cells were then collected bybrief centrifugation.

Plating in BGM after the 3.5 day EB dissociation step was denoted as day0 of erythroid culture. The time period for the entire procedure was19-21 days from the plating of EB cells in BGM medium, with a finalculture volume of 3-4 liters for 5-6×10⁶ MA01 hESCs. It was observedthat the efficiency of RBC generation from MA99, H1 and HuES-3 wasapproximately 5-6 times less than from MA01 hESCs (with acorrespondingly lower final culture volume). RBCs obtained from thisprocedure (before put into culture for further maturation andenucleation) were used for functional characterization, flow cytometryand hemoglobin analyses. The large scale culture experiments werecarried out with hESC lines MA01 (n=6), H1 (n=2), HuES-3 (n=2), and MA99(n=1).

For further maturation, cells collected at day 18-19 (step 3) werediluted with IMDM containing 0.5% BSA (1:5 dilution) and centrifuged at450g for 10 min. To partially enrich the cells for RBCs, the top whiteportion of cell pellet was removed using a pipette with a long fine tip.The RBCs were then plated in Stem Pro-34 SCF (Invitrogen) mediumcontaining SCF (100 ng/ml) and Epo (3 unit/ml) at a density of 2×10⁶cells/ml. The cells were cultured 6 days with media changes every 2days, and then switched to StemPro-34 containing Epo (3 unit/ml) for 4-5more days. These cells were used for β-globin chain and benzidine stainanalyses.

FACS Analysis of Erythroid Cells

All of the conjugated antibodies and the corresponding isotype controlswere purchased from Pharmingen/BD Biosciences except for the RhD and HbFassay (ComDF) purchased from Chemicon. The antibodies used were HLAabc,Duffy group, CD14, CD15, CD34, CD35, CD36, CD41, CD44, CD45, CD71,CD133, CD184 (CXCR4), GPA, RhD and HbF. Erythroid cells were collectedat 19-21 days and washed 2× in PBS with 0.1% BSA and stained inaccordance with the manufacturer's suggested concentration of conjugatedantibody for 30 min at 4° C. The stained cells were then washed 2× inPBS+0.1% BSA and fixed with the wash buffer supplemented with 1%paraformaldehyde. The RhD and HbF assay was performed per manufacturer'sprotocol that included a 0.5% glutaraldehyde/0.1% BSA in PBS prefixingtreatment and a 0.1% Triton X/0.1% BSA in PBS permeabilization stepprior to staining.

After staining with the ComDF reagent for 15 min at room temperature,cells were washed 1× in 0.1% BSA in PBS and fixed in wash buffersupplemented with 1% paraformaldehyde. The samples were then analyzedusing a flow cytometer (FacScan, Becton Dickinson). Cell populationswere analyzed with the CellQuest program (Becton Dickinson)

Functional Analysis of Hemoglobin

Cells collected at 19-21 days were washed 3 times in 0.9% NaCl, thensuspended in 9 volumes of water, lysed with saponin, and clarified bycentrifugation at 600×g. Hemoglobins were then separated by celluloseacetate electrophoresis. Oxygen equilibrium curves were determined usinga Hemox-Analyzer, Model B (TCS Scientific Corp., New Hope, Pa.). The gasphase gradients were obtained using nitrogen and room air, and thecurves were run in both directions. Data were used only from runsshowing negligible hysterisis as described previously (Honig et al., Am.J. Hematol. 1990; 34:199-203; Honig et al., J. Biol. Chem. 1990;265:126-132). Globin mass spectra were obtained using a Voyager-DE ProMALDI-TOF mass spectrometer (Applied Biosystems, Foster City, Calif.) asdescribed by Lee at al. (Rapid Commun. Mass Spectrom. 2005;19:2629-2635). In brief, ZipTips (Millipore, Billerica, Mass.) packedwith C18 and C4 resin were used to prepare the solution for MS analysisof peptide and protein, respectively. Cyano-4-hydroxycinnamic acid(CHCA) and sinapinic acid (SA) were used as the matrix for peptide andprotein, respectively. Aliquots (1.3 ml) of the matrix solution (3-10 mgCHCA or SA in 1 ml aqueous solution of 50% acetonitrile containing 0.1%TFA) were used to elute the peptide/protein from ZipTips and spottedonto a MALDI-TOF (matrix-assisted laser desorption/ionizationtime-of-flight) target. A Voyager-DE PRO Mass Spectrometer (AppliedBiosystems) equipped with a 337 nm pulsed nitrogen laser was used toanalyze the samples. Protein mass was measured using the positive-ionlinear mode. External mass calibration was performed using the peaks ofa mixture of cytochrome c (equine) at m/z 12362, apomyoglobin (equine)at m/z 16952, and adolase (rabbit muscle) at m/z 39212.

RhD and ABO Genotyping

RhD genotyping of hES cell lines by PCR was reported by Arce at al.(Blood 1993; 82:651-655) and Simsek et al. (Blood 1995; 85:2975-2980)with minor modifications. Since all hES cells were maintained on MEF,the inventors designed a pair of human DNA specific PCR primers thatonly amplified human DNA sequences. Genotyping of ABO blood group wasdeveloped based on the polymorphism of glycosyltransferase among ABOblood group individuals (Yamamoto et al., Nature 1990; 345:229-233).

Characterization of hESC-Derived Erythroid Cells

Cells collected at different time points were cytospun at low speed(<1000 rpm) on superfrost plus slides (VWR). Slides were dried andstained with Wright-Giemsa dye for 5 min and washed three times withdistilled water. For immunofluorescence staining, cytospun slides werefixed in 4% paraformaldehyde for 15 min, incubated in 1% BSA for 30 minand incubated overnight at 4° C. in 1:200 primary antibodies ofCD235a/Glycophorin A (Dako), CD71 (BD Biosciences), or human β-globinchain specific antibody (Santa Cruz Biotechnology). Cells were thenincubated for 1 h in 1:200 secondary anti-mouse IgG conjugated torhodamine or FITC (Jackson ImmunoResearch Lab). For total hemoglobinstain, cells at different stages of differentiation using the erythroidexpansion maturation protocol outlined above were collected and cytospunon slides. Air dried cytospin samples were fixed in 100% methanol for 10min. After washing with PBS for 10 min, cells were stained with3′3-diaminobenzidine reagent (Sigma) according to manufacturer'sinstruction. The cells (like all RBCs) containing hemoglobin stainedbrown and nuclei of cells stained blue with Wright-Giemsa.

For immunological blood type characterization, erythroid cells werecollected at 19-21 days, cytospun on glass slides and stained withmonoclonal anti-human blood group A and B antibodies (Virogen, MA)overnight at 4° C. Slides were then incubated with correspondingsecondary antibodies labeled with Rhodamine or FITC (JacksonImmunoResearch Lab) for 30-60 min. After a final wash, the cells werechecked by fluorescence microscopy.

RT-PCR Analysis

Erythroid cells differentiated at different stages using the erythroidexpansion protocol outlined above were collected and the expression ofβ-, γ- and ε-globin genes was analyzed by RT-PCR. In brief, total RNAwas isolated using an RNAeasy Micro Kit (Qiagen), cDNA pools wereconstructed using the SMART cDNA synthesis kit (Clontech) as previouslyreported (Lu et al., Blood 2004; 103:4134-4141). Primers specific forβ-, γ- and ε-globin genes, as reported previously (Qiu et al., Blood2008; 111:2400-2408), were used to amplify corresponding messages. PCRproducts were separated on a 2.5% agarose gel and visualized by ethidiumbromide fluorescence.

Enucleation of hESC-Derived Erythroid Cells In Vitro

Blast cells were cultured as described above up until day 7.

Step 1: Day 7 blast cells in BGM were filtered and plated in Stemline II(Sigma) with supplements based on Giarratana et al. (Nat. Biotechnol.2005; 23:69-74). These included 40 μg/ml inositol, 10 μg/ml folic acid,160 μM monothioglycerol, 120 μg/ml transferrin, 10 μg/ml insulin, 90ng/ml ferrous nitrate, 900 ng/ml ferrous sulfate, 10 mg/ml BSA (StemCell Technologies), 4 mM L-glutamine (Gibco), and 1%penicillin-streptomycin (Gibco). All reagents were from Sigma unlessotherwise noted.

Step 2: For the first seven days in this media (day 7-14), cells werecultured in 1 μM hydrocortisone, 100 ng/ml SCF (Invitrogen), 5 ng/ml IL3(Invitrogen) and 3 IU/ml Epo (Cell Sciences) and maintained at 1×10⁶cells/ml.

Step 3: From day 14 onward, SCF and IL3 were discontinued and Epo wascontinued. Cells were maintained at a density of 2×10⁶ cells/ml. Mediumwas changed every few days.

Step 4: Cells were co-culture with human mesenchymal stem cells (MSC,Lonza) or OP9 mouse stromal cells at various time points (day 19-36) inStemline II with supplements described above and Epo. Before co-culture,MSCs were expanded in MSC Growth Medium (MSCGM, Lonza) and OP9 cellswere expanded in 20% FBS (Atlas) in α-MEM (Invitrogen) with 4 mML-glutamine and 1% penicillin-streptomycin (Gibco).

Statistical Analysis of Cell Dimensions

The area of cells and nuclei on cytospun Wright-Giemsa stained slideswere measured during the enucleation protocol using Scion Image. Thearea of the cytoplasm was calculated as the difference between the totalcell area and nuclear area and nuclear to cytoplasmic ratio (N/C).Diameter was calculated from the area of the nucleus. Differencesbetween diameter and N/C at each time point were measured by an analysisof variance (ANOVA), followed by the Holm's test. Data was presented asmean+/−standard deviation with significance of at least P<0.05.

Example 2 Differentiation of hESCs into Red Blood Cells

Blast cells (BCs) were generated from hESCs as previously described (Luet al., Nat. Methods 2007; 4:501-509). A four-step protocol was employedto differentiate the BCs toward the erythroid lineage, which included[1] EB formation from undifferentiated hESCs, [2] BC formation andexpansion, [3] erythroid differentiation and amplification into a masspopulation of red blood cells and [4] enrichment of red blood cells.Early-stage EBs were generated from hESCs cultured in serum-free mediasupplemented with a combination of morphogens and early hematopoieticcytokines. The EBs were then dissociated and individual cells wereplated in serum-free semi-solid blast-colony growth medium (BGM) for thegrowth and expansion of BCs. Grape-like blast colonies appeared at thebeginning of 3 days, and rapidly expanded from 4 days. The BCs were theninduced to proliferate and differentiate into erythrocytes by adding BGMand Epo for several days. To further expand the erythroid cells,Stemline II-based media containing SCF, Epo, and methylcellulose wasadded every 2 or 3 days for one week. Cells were then diluted in IMDMwith added BSA, collected by brief centrifugation and plated in tissueculture flasks overnight to allow the non-erythroid cells to attach. Theremaining non-adherent cells were collected (representing greater than95% erythroid cells) (FIGS. 1A, 1B, 1C and 1D). Using this optimized(19-21 day) protocol of expansion and differentiation with the additionof bFGF (20 ng/ml) and HOXB4 protein (2 μg/ml) in BGM medium,3.86±1.19×10¹⁰ (mean±SD, n=6) RBCs were generated from one 6-well plateof MA01 hESCs (≈1.2×10⁷ cells). RBCs were also generated with highefficiency from H1 (n=2), HuES-3 (n=2), and MA99 (n=1) hESCs, but theyield was 5-6 times less that obtained from MA01 hESCs. The inventorsfound that the quality of hESCs is one of the most important factors forhigh-efficient generation of RBCs; high quality hESCs (i.e., hESCculture should be composed of colonies with tight borders with minimalsigns of differentiation as seen under microscope at about 80% confluentbut not touching each other; grown at moderated rate: 1:3 split gettingconfluent in 3-5 days; stained positive with markers of pluripotency foralmost every cells; and formed uniform EBs 24 hours after replating)usually generate a high number of EB cells (e.g., 2×10⁶ high qualityhESCs will generate≈2-3×10⁶ EB cells after 3.5 days). It was also notedthat the presence of 0.2-0.5% methylcellulose in the differentiation andexpansion medium prevents cells from aggregating, resulting in enhancedexpansion.

Example 3 Characterization of hESC-Derived RBCs

Morphologically, the RBCs obtained using the above (19-21 day) protocolwere nucleated (>95%) and substantially larger than definitiveerythrocytes with an average diameter of approximately 10 μm.Giemsa-Wright staining showed an abundance of hemoglobin in thecytoplasm (FIGS. 1C and 1D). The identity of the cells was confirmed byimmunological characterization (Table 1 and FIG. 1F). Over 65% of thecells expressed fetal hemoglobin (HbF), >75% were CD71 positive, and 30%of the cells expressed CD235a, whereas the majority of the cells did notexpress myelomonocytic or megakaryocytic antigens (All cells werenegative for CD14, whereas 0.4% of cells expressed CD15; 8.6% of cellsexpressed CD41) and progenitor antigens (0.3% cells were positive forCD34; 10% cells expressed CD35, and 5% cells were positive for CD36)(Table 1). The inventors have previously shown that BCs express thechemokine receptor CXCR413. However, the inventors did not detect theexpression of CXCR4 or CD133 on the surface of the hESC-derived RBCs,which is consistent with the findings from erythroid cells expanded fromcord blood progenitors in vitro (Giarratana et al., Nat. Biotechnol.2005; 23:69-74; Miharada et al., Nat. Biotechnol. 2006; 24:1255-1256).Interestingly, few or none of the cells expressed HLA (<5%) or Duffy(0%) group antigens, a finding that has also been observed forCD34+CD38− hematopoietic precursors derived from hESCs (Lu et al., Blood2004; 103:4134-4141).

Mass spectral analysis showed that the main globin types found in theRBCs obtained at day 19-21 from MA01 and H1 hESCs included the embryonicζ- and ε-chains, and the fetal Gγ-chain (FIG. 1E). Substantialquantities of α-chains were also present, but neither Aγ- nor adultβ-globin chains could be detected. Nevertheless these resultsdemonstrate that hemoglobin synthesis in these cells corresponds to theembryonic and early fetal developmental stage, and are consistent withrecent reports showing that even definitive-appearing erythroid cellsderived from hESCs coexpress high levels of embryonic and fetal globinswith little or no adult globin (Lu et al., Blood 2004; 103:4134-4141;Chang et al., Blood 2006; 108:1515-1523; Qiu et al., Blood 2008;111:2400-2408; Lu et al., Stem Cells Dev. 2007; 16:547-560).

Example 4 Functional Analysis

In six separate experiments, the oxygen equilibrium curves of thehESC-derived erythroid cells (day 19-21 cultures) were either verysimilar to (FIG. 2A) or somewhat rightward shifted, relative to that ofnormal adult RBC's. The oxygen equilibrium curve illustrated in FIG. 2Ahas a biphasic appearance. At the low end of the oxygen saturation, itscurve is to the left of the normal, and it is hyperbolic in shape(arrow). At their midpoint, the two curves are virtually identical, andat higher saturation levels, the curve of ESC-derived erythroid cells isagain displaced slightly to the left of the normal (arrow head). Hill'sn coefficient was also similar to that of the normal control (FIG. 2C).The ESC-derived erythroid cells showed a comparable Bohr effect atphysiological and higher pH values, but a lesser shift at lower pH (FIG.2B). The response to 2,3-diphosphoglycerate (2,3-DPG) depletion of thesecells was significantly less than in the normal control (FIG. 2C),consistent with the known lack of interaction between Hb F and 2,3-DPG(Maurer et al., Nature 1970; 227:388-390). These findings demonstratethat the hESC-derived RBCs have oxygen carrying properties that arecomparable to those of normal adult erythrocytes.

Example 5 Generation of RhD(−) RBCs from hESCs

The manufacture of O/RhD(−) RBCs would substantially aid in theprevention of alloimmunization when transfused into RhD(−) mismatchedpatients. The anticipated need for universal donor RBCs (O—) in Westerncountries is greater than in Asian countries such as Korea, Japan andChina, where the RhD(−) type is less prevalent (<0.5% vs 15%,respectively). Genotype analysis by PCR showed that only two out oftwenty hESC lines studied, MA99 and MA133, were RhD(−) (FIG. 3A).Erythroid cells from 19-21 day cultures were used for FACS andimmunological analyses. FACS analyses demonstrated that RBCs generatedfrom MA01 expressed RhD antigen on their surfaces, whereas cells derivedfrom MA99 lacked the expression of RhD antigen (FIG. 3D), confirming theresults of genomic DNA PCR analysis (FIG. 3A). Immunocytochemicalanalysis using monoclonal antibodies against the A and B antigens showedthat approximately 5% of RBCs generated from MA01 cells expressed the A,but not the B antigen (FIG. 3E), demonstrating that MA01 cells have aphenotype of A(+); about 5% of RBCs derived from MA99 cells expressedthe B, but not the A antigen (FIG. 3E), suggesting MA99 cells have aB(−) phenotype, while RBCs derived from WA01 cells expressed neither Anor B antigens, confirming WA01 cells as O-type, consistent with theresults of genomic PCR analysis (FIGS. 3B and 3C). However, it is worthnoting that not all erythroid cells expressed the A or B antigen, whichmay reflect the early developmental stage of the cells (Wada et al.,Blood 1990; 75:505-511; Hosoi et al., Transfusion 2003; 43:65-71).

Example 6 Enucleation and Maturation of hESC-Derived Erythroid Cells InVitro

A critical scientific and clinical issue is whether hESC-derivederythroid cells can be matured in vitro to generate enucleatederythrocytes. To investigate this, several different strategies andculture conditions were studied. It was found that hematopoietic stemcell expansion medium Stemline II plus supplements and cytokinesreported by Giarratana et al. (Nat. Biotechnol. 2005; 23:69-74)supported the growth, expansion, maturation and enucleation ofhESC-derived erythroid cells with significantly higher efficiency thanother tested conditions. Blast cells cultured in this condition withoutstromal layers resulted in 10-30% enucleation, while culturing on MSCstromal cells resulted in approximately 30% enucleation and OP9 stromalcell layers further enhanced the enucleation process. Approximately30-65% of erythroid cells (40±17% [mean±SD, n=4]) were enucleated whenthese cells were transferred to OP9 stromal layers from non-stromal fiveweek cultures and co-cultured from days 36-42 (FIGS. 4C and 4E). Theenucleated erythrocytes (FIGS. 4C and 4E) show similar staining patternand size as mature RBCs from normal human blood (FIGS. 4D and 4F). Theseerythroblasts were derived from hESCs grown without MEFs using the BDMatrigel system. The fact that erythroblasts kept in non-stromalconditions (without transfer to MSC or OP9) could enucleate 10-30%suggests that enucleation could be achieved completely feeder-free.

Total of six experiments were performed with hESC lines H1 (n=3), MA01(n=2) and huES-3 (n=1), all exhibiting varying levels of enucleation andexpansion of 30-50-fold. Stromal cells, especially OP9, were able toenhance survival of the cells after long term culture compared tonon-stromal conditions.

To further investigate the events associated with enucleation, multiplecharacteristics related to the process of erythrocyte maturation wereexampled. It was observed a progressive decrease in cell size andnuclear to cytoplasm (N/C) ratio before enucleation occurred. Prior totransfer to the OP9 stromal layer, the size and N/C of these cellsdecreased significantly from 18.3 μm in diameter on day 8 to 12.9 μm fornucleated cells (p<0.001) and to 7.5 μm for enucleated cells on day 27(p<0.001), and N/C ratios from 0.82 on day 8 to 0.30 by day 27 (p<0.001,FIGS. 4A and 4B), indicating substantial nuclear condensation during theprocess. Wright-Giemsa stains demonstrated a gradual progression fromblue to purple to pink stain, indicative of pronormoblast topolychromatic erythroblast to orthochromatic normoblast transition.These cells expressed a high level of CD71, an early erythroblastmarker, on day 8 and decreased their expression over time; whereas theyshowed low to negligible level of CD235a (Glycophorin A) protein, amature erythrocyte marker, in the beginning, but increased theirexpression dramatically with their maturation (FIG. 5A and FIG. 6).Benzidine stains also showed a progressive accumulation of hemoglobinsin these cells and a decrease in cell size over time (FIG. 5C).

Preliminary experiments confirmed that the immature enucleated erythroidcells mainly expressed the embryonic ζ- and ε-globin chains, and thefetal γ-globin chain (FIG. 1E). Although substantial quantities ofα-chains were present in these cells, adult β-globin chains were notdetected. Subsequent studies were carried out to determine whether theerythroid cells possess the capacity to express the adult definitiveβ-globin chain upon further differentiation and maturation in vitro.Globin chain specific immunofluorescent analysis showed that the cellsincreased expression of the adult β-globin chain (0% at day 17, FIG. 5B)to about 16.37% after 28 days of in vitro culture (some cells expressedthe β-globin chain at very high levels, FIG. 5B and FIG. 7). Theexpression of p-globin chain gene in these cells was confirmed by globinchain specific RT-PCR analysis (Qiu et al., Blood 2008; 111:2400-2408)(FIG. 8). Consistent with a recent report (Zambidis et al., [abstract].6th ISSCR Annual Meeting 2008; 357), the inventors also observed thatall the cells expressed the fetal γ-globin chain irrespective of theβ-globin chain expression status.

TABLE 1 Characterization of hESC-derived erythroid cells by FACSanalysis Antibodies Positive Range (%. n = 5) Average (Mean ± SE) HbF40.03-96.60 66.79 ± 9.88 CD47 95.00-99.21 97.51 ± 0.85 GPA 21.31-41.9330.10 ± 3.79 CD71 59.40-83.39 76.07 ± 4.33 CD44 18.61-44.56 30.72 ± 4.55CD45 10.06-40.21 22.23 ± 5.45 CD41  4.44-20.16  8.61 ± 2.98 CD14 0 0CD15 0.20-0.60 0.38 ± 0.08 CD34   0-1.62 0.34 ± 0.32 CD35  5.82-17.469.79 ± 2.00 CD36  1.08-13.30 4.99 ± 2.14 CD133 0 0 CD184 (CXCR-4) 0 0Duffy 0 0 HLAabc 0.75-6.25 4.15 ± 1.14

Example 7 RhD and ABO Genotyping

RhD genotyping of hES cell lines by PCR was reported by Arce et al. andSimsek et al. (Arce et al., Molecular cloning of RhD cDNA derived from agene present in RhD-positive, but not RhD-negative individuals. Blood1993; 82:651-655; Simsek et al. Rapid RhD genotyping by polymerase chainreaction-based amplification of DNA. Blood 1995; 85:2975-2980) withminor modifications. Since all hES cells were maintained on MEF, theinventors designed a pair of human DNA specific PCR primers that onlyamplified human DNA sequences PCR primers were: RhD-F,5′-tgaccctgagatggctgtcacc-3′ (SEQ ID NO: 34) and RhD-R,5′-agcaacgatacccagtttgtct-3′ (SEQ ID NO: 35), which amplify intron 4between exons 4 and 5, and generate only a 1,200 by fragment with DNAfrom RhD negative individuals, whereas in RhD positive individuals, 100bp and 1,200 bp (which is weak due to the fragment size ofamplification) are generated. This strategy has been confirmed to be incomplete agreement with serologically determined phenotypes (Simsek etal., Blood 1995). In brief, genomic DNA was isolated from hES cellsusing a QIAamp DNA Mini Kit (Qiagen, Valencia, Calif.), and 200 ng DNAper reaction in 50 μl was used for PCR amplification. PCR conditions:94° C. for 45 sec, 60° C. for 1.5 min, and 72° C. for 2.0 min for 35cycles with final extension at 72° C. for 7 min. PCR products wereseparated on a 1.2% agarose gel and visualized by ethidium bromidestaining. DNA from mononuclear cells of normal human blood with RhDpositive and negative individuals was used as positive and negativecontrols.

Genotyping of ABO blood group was developed based on the polymorphism ofglycosyltransferase among ABO blood group individuals (Yamamoto et al.,Molecular genetic basis of the histo-blood group ABO system. Nature1990; 345:229-233.). First, human specific PCR primers were designed toamplify a DNA fragment surrounding nucleotide 258, in which O allelecontains one nucleotide (G) deletion at this site and generates acutting site for restriction enzyme Kpn I, but eliminates a cutting siteof restriction enzyme Bst EII. PCR products were then subjected torestriction digestion by Kpn I and Bst EII: PCR product from O/Ogenotype can only be digested by Kpn I to generate two new shorterfragments, but is resistant to the digestion of Bst EII; while PCRproduct from A/A, B/B and A/B genotypes is resistant to Kpn I digestion,and is only cut by Bst EII; whereas PCR product from genotypes of A/O orB/O can be digested partially by both enzymes. Therefore, the first PCRamplification and restriction digestion is able to distinguish O bloodtype and non-O blood type. Based on the results, the second set of PCRprimers were designed to amplify the region of nucleotide 700, whereboth A and O alleles contain a G nucleotide that can be digested by MspI, while the B allele has an A nucleotide at this position thatgenerates an Alu I cutting site. The combination of two separate PCRamplification at two diagnostic positions of the glycosyltransferase,and four restriction enzyme digestions can clearly distinguish A, B or Oalleles. In brief, the PCR reaction was carried out with a set ofprimers amplifying the region of nucleotide 258 (primers: O-type-F,5′-gccgtgtgccagaggcgcatgt-3′ (SEQ ID NO: 36), O-Type-R,5′-aatgtccacagtcactcgccac-3′ (SEQ ID NO: 37), PCR product, 268 bp), thePCR product was purified by a Qiagen Kit, digested by Kpn I and Bst E11,and separated on a 2% agarose gel and visualized by ethidium bromidestaining. For the O/O genotype, Kpn I generates 174 bp and 93 bpfragments, and Bst EII does not cut the PCR product; for the A/A, B/Band A/B genotypes, Kpn I does not cut the PCR product, Bst EII generates174 bp and 93 bp fragments; for A/O or B/O genotypes, both Kpn I and BstEII partially cuts the PCR product and generates 267 bp (original), 174bp and 93 bp fragments. Second PCR amplification using primersamplifying the region of nucleotide 700 was carried out (primers:AB-Type-F, 5′-tgctggaggtgcgcgcctacaag-3′ (SEQ ID NO: 38), AB-Type-R,5′-gtagaaatcgccctcgtccttg-3′ (SEQ ID NO: 39), PCR product, 278 bp), PCRproduct was purified, digested by Alu I and Msp I and separated asabove. For the B/B genotype, Alu I digestion generates 187 bp+91 bpfragments, and Msp I digestion generates 206 bp+47 bp. For A/A, A/O andO/O genotypes, Alu I does not cut the PCR product, Msp I generates 187bp+47 bp fragments. For the A/B or B/O genotypes, Alu I generates 278 bp(no cut)+187 bp+91 bp fragments; and Msp I generates 206 bp and 187bp+47 bp fragments.

Example 8 Materials and Methods

Culture of hESCs

hESC lines WA01(H1), HUES3, and MA01 were used and maintained aspreviously described⁽⁶⁾. Briefly, hESCs were grown on mitomycinC-treated mouse embryonic fibroblast (MEF) in complete hESC media. ThehESCs were passaged every 3-5 days before reaching confluence using0.05% trypsin-0.53 mM EDTA. For feeder-free culture, the cells were thengrown on hESC-qualified Matrigel matrix (BD Biosciences) in completeModified TeSR™1 (mTeSR™1) medium (Stem Cell Technologies, Inc), which isbased on the formulation of Ludwig et al.^((7,8)). Cells were maintainedaccording to manufacture's suggested instructions. Briefly, cells werepassaged when they reached approximately 90% confluence, usually every5-7 days with split ratios ranging from 1:3 to 1:6. Cells were treatedwith dispase (1 mg/ml BD, Biosciences) and incubated for 3-5 minutes at37° C. to begin dislodging the colonies. Colonies were washed withDMEM/F12 (Mediatech) to remove dispase solution. To extricate thecolonies from the tissue culture plastic, the wells were coated withDMEM/F12 and gently scraped until all of the colonies had beendisplaced. The colonies were transferred to conical tubes, the wellswere washed with DMEM/F12 and the cells pooled to collect any remainingin the wells. They were centrifuged for 5 minutes at 1000 rpm. The cellpellets were resuspended in mTeSR™1 media and transferred to Matrigelcoated 6 well plates, in 2 ml of mTeSR™1 media per well. Cells weremaintained at 37° C. under 5% CO2 and the mTeSR™1 medium was replenisheddaily.

Immunofluorescent Cytochemistry Analysis

Feeder-free hESC colonies were assayed for Oct-4 and Tra-1-60 expressionusing immunofluorescence. The cells were fixed with 4% paraformaldhyde(PFA), washed with PBS, and blocked with 5% Normal Goat Serum (VectorLabs), 1% BSA (Sigma) and 0.2% Triton-X-100 (Sigma) in PBS for 30minutes at room temperature. Cells were incubated with primaryantibodies against Oct-4 (Santa Cruz Biotechnology) or Tra-1-60(Millipore/Chemicon), in blocking solution, overnight at 4° C., washedwith PBS and incubated with a biotin conjugated secondary antibody(Jackson ImmunoResearch Labs), in blocking solution, for 45 minutes atroom temp. After further washing, cells were incubated with Alexa 954conjugated streptavidin (Invitrogen/Molecular probes), for 15 minutes atroom temp followed by an extended final wash in PBS. Cells were mountedin Prolong Gold with DAPI (Invitrogen/Molecular Probes).

Differentiation of Hemangioblasts from hESCs

To induce hESCs cultured on MEFs into hemangioblasts, 80-90% confluentplates were dissociated by 0.05% trypsin digestion. To differentiatefeeder-free hESCs into hemangioblasts, 85-90% confluent cells weredislodged from the Matrigel matrix using the protocol described above.Cells from both conditions were plated on Ultra-Low dishes (Corning,N.Y.) in Stemline II (Sigma) medium with different doses of BMP-4, VEGFand bFGF as described previously⁽²⁾. Half of the medium was replacedafter 48 hours with fresh medium containing the same cytokines or thesame medium plus SCF, FLT3 ligand (FL) and Tpo (20 ng/ml, R&D System)which depend on different experiment conditions. After 3.5 days, EBswere collected and dissociated by 0.05% trypsin. Single-cell suspensionswere obtained by passing the cells through 22-gauge needle and through a40-μm cell strainer, collected by centrifugation, and resuspended in50-100 μl of Stemline II media. Cells (0.75×10⁵ to 1×10⁵) were mixedwith 2.5 ml of blast colony growth medium (BGM) as previouslydescribed⁽²⁾, plated in Ultra-Low dishes and incubated at 37° C. Blastcolonies derived from both MEF and feeder-free hESCs were observed 3-4days after plating, followed shortly thereafter by rapid expansion.Blast cells (BC) are defined in the current study as cells obtained fromday-6 blast colonies.

Enrichment of Hemangioblast Precursors

Potential BC precursor surface markers CD31, CD34, KDR, CXCR-4, CD133,ACE, PCLP1, PDGFRα, Tie-2, Nrp-2, Tpo-R and bFGFR-1 were selected forcell enrichment. All antibodies are mouse monoclonal IgG isotype andthey are: CD31 and CD34 (Dako Cytomation), KDR and Tpo-R(R&D Systems,Inc.), CXCR-4 (Abcam Inc.), Nrp-2, ACE, PCLP1 and PDGFRα (Santa CruzBiotechnology), Tie-2 (Cell Signaling Technology, Inc.), bFGFR-1 (ZymedLaboratories), and CD133 (Miltenyi Biotech). Antibody cocktail assemblywas performed by EasySep “Do-it-Yourself” Selection Kit (Stem CellTechnologies). Cell suspensions derived from EBs were centrifuged at1200 rpm for 4 min and resuspended in PBS with 2% FBS/1 mM EDTA bufferat a concentration of 1-2×10⁶ cells/100 μl. The cells were mixed withdifferent antibody cocktails for 15 min at RT and then incubated withEasySep Nanoparticle at RT for 10 additional minutes. Positive selectedcells were separated after pouring off supernatant when placing tubewith cells in a Magnet holder. Antibody selected positive cells (1×10⁵)were mix with 2.5 ml of BGM and plated for blast colony development.

Real Time RT-PCR and Data Analysis

Total RNA was extracted from EBs or undifferentiated hESCs using RNeasyMicro Kits (Qiagen) according to manufacture's protocol. cDNAs weresynthesized using BD SMART PCR cDNA Synthesis Kit (BD Biosciences) permanual instructions. Real time RT-PCR (qRT-PCR) was performed usingFullVelocity SYBR Green QPCR Master Mix (Stratagene). The reactions wereset up in triplicate with the following components per reaction: 50 ngof template, 0.2 micromoles of each primer and 1× Master mix. Genespecific sequences of the primers used are listed in Table 1, andannealing temperature for all primers is 55° C. Amplification andreal-time data acquisition were performed in a Stratagene Mx3005P withMxPro version 3.0 software. The following cycle conditions were used:one cycle of 95° C. for ten minutes followed by forty cycles of 95° C.for 30 seconds, 55° C. for 1 minute, 72° C. for 30 seconds followed by afinal cycle of 95° C. for 1 minute, 55° C. for 30 seconds and 95° C. for30 seconds. Relative quantification of each target gene was performedbased on cycle threshold (C_(T)) normalization to β-actin (ΔC_(T)) usingthe ΔΔC_(T) method⁽⁹⁾. Analysis of relative gene expression data usingreal-time quantitative PCR and the 2(-delta deltaC(T)) method⁽⁹⁾, wherethe ΔC_(T) of each examined gene in the experimental samples wascompared to average ΔC_(T) of each gene in an undifferentiated hESCcontrol sample (ΔΔC_(T)). Then the fold change in expression wascalculated as 2^(−ΔΔCT). The negative fold difference data was convertto a linear “Fold change in expression” value using the followingformula: Linear Fold Change in expression=−(1/fold change inexpression).

Statistical Analysis

All data were presented as mean±SEM. Intergroup comparisons wereperformed by unpaired Student's t-test using GraphPad Prism, version 4,software (GraphPad Software, Inc., San Diego, Calif.). p<0.05 wasinterpreted as statistically significant.

Example 9 Both BMP-4 and VEGFs are Required for HemangioblastDevelopment

A serum free system to induce hESC differentiation toward thehemangioblastic and hematopoietic lineages was previouslydescribed^((2,10)). Although BMP-4, VEGF, and a cocktail of earlyhematopoietic cytokines were used, the absolute requirement and optimalconcentrations of the individual factors were not examined. In order toreduce the expense and effort necessary to generate hemangioblasts forfuture research and clinical applications, the inventors specificallyexamined the minimal requirements and effects of VEGFs, BMPs, and threeearly hematopoietic cytokines (TPO, FL and SCF) on the efficientdevelopment of blast colonies from hESCs. It was found that BMP-4 isabsolutely required for the development of blast colonies underserum-free conditions. No blast colonies were obtained without thesupplement of BPM-4 in the medium during EB formation and a cleardose-response effect of BMP-4 was observed for the formation of blastcolonies from hESCs (FIG. 9A). Furthermore, BMP-4 could not besubstituted by other members of the BMP family. BMP-2 and BMP-7 alone,or a combination of the two, failed to promote BC development.Furthermore, supplementation of BMP-2 and BMP-7 in EB medium containingBMP-4, either showed no effect (10 ng/ml) or inhibited (20 ng/ml) blastcolony development (FIG. 9B). However, addition of BMP-4, and BMP-2and/or BMP-7 in blast colony growth medium (BGM) did not have any effecton the development of blast colonies, suggesting that BMP-4 onlypromotes the mesoderm/hemangioblastic specification stage, but not thegrowth and expansion of BCs. Similarly, no blast colonies developed whenVEGF₁₆₅ was eliminated from the EB formation medium. VEGF₁₆₅ was foundto promote the development of blast colonies in a dose dependent manner(FIG. 9C). VEGF₁₂₁, an isoform of VEGF members that can only bind to KDRand FLT1 receptors⁽¹¹⁾, can be used as a substitute of VEGF₁₆₅ inpromoting the development of blast colonies from hESCs; almost identicalnumbers of blast colonies (68±5 vs. 67±12) were developed when 50 ng/mlof either VEGF₁₆₅ or VEGF₁₂₁, which is the optimal dose under serum-freecondition, was added in EB medium. However, in contrast to BMP-4, noblast colonies were obtained if VEGF was absent in BGM, demonstratingthat VEGF plays a critical role both in early stage ofmesoderm/hemangioblastic specification and in the growth and expansionof BCs.

In the inventors' original report⁽²⁾, TPO, FL and SCF were added 48hours after plating hESCs in EB medium in an effort to further promoteearly hematopoietic progenitor growth and expansion. Here it wasexamined whether TPO, FL, and SCF played any role in the specificationof hESCs toward the mesoderm/hemangioblast lineage. EBs were formed byplating hESCs in Stemline II medium with 50 ng/ml of BMP-4 and VEGF, anddivided into two wells after 48 hours: to one well, 20 ng/ml of TPO, FLand SCF was added, to the other well, no additional factor was added,and the EBs were incubated for another 36 hours. EBs were then collectedand single cell suspension was obtained and plated for blast colonyformation. Our results show that supplement of TPO, FL and SCF during EBformation has no effect on the development of blast colonies, 242±16 vs.287±33 blast colonies developed per 1×10⁵ cells derived from EBs treatedwith and without TPO, FL and SCF, respectively.

Example 10 bFGF Promotes the Growth, but not Commitment, ofHemangioblasts from hESCs

Previous studies have shown that supplement of bFGF during earlydifferentiation promotes murine and human ESC hematopoieticdevelopment^((12,13,14,5)). Thus, we investigated whether the additionof bFGF during the EB differentiation stage would enhance blast colonyformation from hESCs. Addition of bFGF during EB formation had no effecton the development of blast colonies, and, in fact, at a higher dose (40ng/ml) inhibited the formation of blast colonies from multiple hESClines (FIG. 10A and FIGS. 11). In contrast, the addition of bFGF in BGMsignificantly enhanced the development of blast colonies (FIG. 10A, FIG.11). Both the number of blast colonies (p<0.001) and total number of BCsincreased significantly compared to BGM without bFGF supplementation.With bFGF at optimal dose (20 ng/ml) in BGM, the blast colonies arelarger and healthier, and we consistently harvest approximately 1×10⁸BCs from one six-well plate of high quality WA01 hESCs (approximately1.2×10⁷ cells) after 6 days growth, which is 8±1 fold higher than thatobtained from BGM without the supplement of bFGF.

To investigate the lineage differentiation potentials of BCs generatedwith and without supplementation of bFGF, equal numbers of pooled BCswere plated for hematopoietic and endothelial lineage differentiation aspreviously described⁽²⁾. For hematopoietic CFU formation, 129±9 and86±22 CFUs/10⁴ BCs were formed from BCs derived from BGMs supplementedwith and without bFGF (20 ng/ml), respectively. Furthermore, nodifference was observed for the development of different CFUs (CFU-mix,CFU-G, CFU-M and CFU-E) between the two groups (data not shown). Forendothelial lineage differentiation, more BCs (62±3%) from BGM with bFGF(20 ng/ml) differentiated into endothelial cells than BCs (55±3%)derived from BGM without bFGF supplement. Endothelial cells from bothsources formed capillary-vascular like structures efficiently afterplating on Matrigel (FIGS. 10B and 2C). These results suggest that bFGFpromotes the growth of BCs, but does not cause preferential lineagedifferentiation.

Example 11 Robust Generation of Hemangioblasts from hESCs MaintainedWithout Feeder Cells

It has been reported that hESCs maintained on MEF feeders contain thenonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc)^((15,7,8)), andthat animal sources of Neu5Gc can cause a potential immunogenic reactionwith human complement. The culturing of hESCs on MEF feeder layersprevents complete elimination of animal Neu5Gc, and raises concerns forthe potential clinical applications of hemangioblasts generated fromhESC lines maintained under these conditions. Therefore, we have takensteps to determine whether hemangioblasts can be generated from hESCsmaintained without MEF feeders. Three hESC lines were passaged withdispase onto plates coated with hESC-qualified Matrigel matrix, andmaintained in mTeSR medium as described in Materials and Methods. Theirundifferentiated state was confirmed with immunofluorescence stainingfor the expression of Oct-4 and Tra-1-60 antigens and colony morphology(FIG. 12A-12H). These cells were collected and utilized for thedevelopment of BCs using the optimized conditions described above.Interestingly, a significantly higher number of BCs were observed withfeeder-free hESCs as compared to hESCs cultured on MEF feeders whenidentical numbers of EB cells were plated (FIG. 12 l, p<0.05). Theseresults were observed for all three tested hESC lines WA01, MA01 andHUES-3 (data not shown).

Example 12 Mechanism Underlying the Effects of BMP-4 and VEGF onHemangioblast Development

In order to dissect the molecular mechanism underlying the effects ofBMP-4 and VEGF on hemangioblast development from hESCs, the inventorscompared the expression of genes associated with the development ofhemangio blasts in 3.5 day-old EBs that were formed in Stemline IImedium both with and without each factor, as well as with a combinationof BMP-4 and VEGF. Gene expression was analyzed by real-time RT-PCR(qRT-PCR) and compared with their levels in undifferentiated hESCs. EBsformed without any factor expressed higher levels of OCT-4, a marker forhESCs, than undifferentiated hESCs. Supplementation of VEGF in EB mediumled to a moderate down regulation of OCT-4 expression; whereas theaddition of BMP-4 or BMP-4 plus VEGF resulted in a significant decreasein OCT-4 expression (p<0.0005, FIG. 13). There was no additive effect ofBMP-4 and VEGF on OCT-4 expression. The expression of T-brachyury gene,the earliest marker expressed in mesoderm cells, was downregulated inall samples except EBs derived from cultures containing both BMP-4 andVEGF (the latter showing a significant increase in its expression(p<0.0005). Similar expression patterns were observed for CD31 and LMO2;significantly increased levels of expression were only detected in EBsexposed to a combination of BMP-4 and VEGF (p<0.0005). KDR, one of themost studied VEGF receptor, has been shown to be expressed in all hESClines^((4,5)); its expression was dramatically down regulated in EBsderived from media with no addition of exogenous factor, and withsupplement of BMP-4 or VEGF alone. However, a moderate but significantincrease in KDR expression was observed in EBs formed in the presence ofBMP-4 and VEGF (p<0.002), a condition that promoted efficientdevelopment of hemangioblasts from hESCs. Surprisingly, in contrast to arecent report⁽¹⁴⁾, substantial decreases in the expression of MixL andSCL/TAL-1 genes were detected in EBs formed in all conditions. Onepossible explanation is that growth in different serum-free media causeda different expression pattern in these genes. Nevertheless, theseresults suggest that the commitment and development ofmesoderm/hemangioblast from hESCs requires both BMP-4 and VEGF,consistent with the results of blast colony development (FIG. 9).

Example 13 Identification of Surface Markers for Progenitors of BlastCells

In our original method⁽²⁾, BCs were generated by replating day 3.5 EBscells in 1% methylcellulose supplemented with defined factors. Thisstrategy is important when identifying BCs that possess the potential toform hematopoietic and endothelial cells, and it is also reproduciblewhen generating BCs from hESCs. However, this approach utilizes dishesin standard tissue culture incubators, and thus cannot be adapted torotary bioreactors for scale-up. This limitation is mainly due to thefact that cells from day 3.5 EBs are heterogeneous and includeundifferentiated hESCs (only a portion of the cells are BC progenitors).Replating this heterogeneous population in liquid culture wouldtherefore lead to the growth of all cells including the formation ofsecondary EBs from undifferentiated hESCs, excluding their possible usein clinical applications. However, if a marker(s) for the progenitor ofBCs can be identified, the purified progenitor can be seeded in liquidculture adapted with a rotary bioreactor for scaled-up production ofBCs. We therefore selected 12 cell surface molecules that are associatedwith the development of mesoderm derivatives. The correspondingantibodies were used to enrich cells from day 3.5 EBs, and the enrichedcells assayed for blast colony forming ability. As shown in FIG. 14,KDR+ cells from 3.5 day EBs generated three times more blast coloniesthan the unfractioned control cells (p<0.01), which is consistent withprevious studies⁽⁵⁾. Although we also found a moderate increase in blastcolonies (≈1.5 fold) after plating CD31+ and CD34+ enriched populations,the increase did not reach statistical significance. All other enrichedpopulations produced equal or less blast colonies as compared withunfractioned control cells, indicating that the BC progenitor does notexpress these molecules. The unbound (flow through) cells of allantibodies tested also formed similar numbers of blast colonies as theunfractioned cells, suggesting that even KDR+, CD34+ and CD31+ cellsrepresent a very limited portion of the cells that are capable offorming blast colonies.

TABLE 1 Sequences of gene-specific primers used in qRT-PCR SEQ ID SEQ IDGene Forward Primer, 5′- 3′ NO Reverse Primer, 5′- 3′ NO Ref OCT-4GAAGGTATTCAGCCAAACGC 16 GTTACAGAACCACACTCGGA 17 NA BRACHTGCTTCCCTGAGACCCAGTT 18 GATCACTTCTTTCCTTTGCAT 19 ⁽³³⁾ CAAG MixL1CCGAGTCCAGGATCCAGGTA 20 CTCTGACGCCGAGACTTGG 21 ⁽³³⁾ KDR/Flk1CCAGCCAAGCTGTCTCAGT 22 CTGCATGTCAGGTTGCAAAG 23  ⁽⁴⁾ CD31GAGTCCTGCTGACCCTTCTG 24 ATTTTGCACCGTCCAGTCC 25  ⁽⁴⁾ Scl/TAL1ATGAGATGGAGATTACTGATG 26 GCCCCGTTCACATTCTGCT 27  ⁽⁴⁾ LMO2AACTGGGCCGGAAGCTCT 28 CTTGAAACATTCCAGGTGATA 29  ⁽⁴⁾ CA GAPDHCGATGCTGGCGCTGAGTAC 30 CCACCACTGACACGTTGGC 31 NA β-ActinGCGGGAAATCGTGCGTGACA 32 GATGGAGTTGAAGGTAGTTTC 33 NA G

Example 14 Generation of Human Hemangio-Colony Forming Cells from HumanES Cells

Human ES cell culture. The hES cell lines used in this study werepreviously described H1 and H9 (NIH-registered as WA01 and WA09) andfour lines (MA01, MA03, MA40, and MA09) derived at Advanced CellTechnology. Undifferentiated human ES cells were cultured on inactivated(mitomycin C-treated) mouse embryonic fibroblast (MEF) cells in completehES media until they reach 80% confluence (Klimanskaya & McMahon;Approaches of derivation and maintenance of human ES cells: Detailedprocedures and alternatives, in Handbook of Stem Cells. Volume 1:Embryonic Stem Cells, ed. Lanza, R. et al., (Elsevier/Academic Press,San Diego, 2004). Then the undifferentiated hES cells were dissociatedby 0.05% trypsin-0.53 mM EDTA (Invitrogen) for 2-5 min and collected bycentrifugation at 1,000 rpm for 5 minutes.

EB formation. To induce hemangioblast precursor (mesoderm) formation,hES cells (2 to 5×10⁵ cells/ml) were plated on ultra-low attachmentdishes (Corning) in serum-free Stemline media (for e.g., Stemline I orII, Sigma™) with the addition of BMP-4 and VEGF₁₆₅ (50 ng/ml, R&DSystems) and cultured in 5% CO2. Approximately 48 hours later, the EBmedium was replenished and supplemented with a cocktail of earlyhematopoietic/endothelial growth factors. For example, half the mediawere removed and fresh media were added with the same finalconcentrations of BMP-4 and VEGF, plus SCF, TPO and FLT3 ligand (20ng/ml, R&D Systems). The triple protein transduction domain (tPTD)-HoxB4fusion protein (1.5 μg/ml) was added to the culture media between 48-72hr to expand hemangioblast and its precursor.

Hemangioblast expansion. After 3.5-5 days, EBs were collected anddissociated by 0.05% trypsin-0.53 mM EDTA (Invitrogen) for 2-5 min, anda single cell suspension was prepared by passing through 22G needle 3-5times. Cells were collected by centrifugation at 1,000 rpm for 5 minutesand counted. Cell pellets were resuspended in 50-200 μl of serum-freeStemline media. To expand hemangioblasts, single cell suspensions fromEBs derived from differentiation of 2 to 5×10⁵ hES cells were mixed with2 ml BL-CFC/hemangioblast expansion media (BGM) containing 1.0%methylcellulose in Iscove's MDM, 1-2% Bovine serum albumin, 0.1 mM2-mercaptoethanol and a cocktail of growth factors. For example, 10μg/ml rh-Insulin, 200 μg/ml iron saturated human transferrin, 20 ng/mlrh-GM-CSF, 20 ng/ml rh-IL-3, 20 ng/ml rh-IL-6, 20 ng/ml rh-G-CSF, 3 to 6units/ml rh-EPO, 50 ng/ml rh-SCF, 50 ng/ml rh-FLt3 ligand, 50 ng/mlrh-VEGF and 50 ng/ml rh-BMP-4) (“rh” stands for “recombinant human”) and1.5 μg/ml of tPTD-HoxB4 fusion protein, with/without 50 ng/ml of TPO andFL was added. The cell mixtures were plated on ultra-low attachmentdishes and incubated at 37° C. in 5% CO₂ for 4-7 days. After 4-6 days,grape-like hemangioblast blast colonies (referred to as BL-CFCs or BCs)were visible by microscopy. Cytospin preparation and Wright-Giemsastaining of the hES-derived blast colonies confirmed morphologicfeatures of immature blast cells. To extend these results to other hEScell lines (WA09 [H9], MA01, MA03, MA40 and MA09, supplements of FL andTpo were necessary for sustained growth of the BC colonies (without FLand Tpo, small (10-20 cell hES-BCs were obtained which died after 4-8days). Epo was also essential for BC formation and growth in all hEScell lines tested. These cells could be readily expanded (one 6-wellplate of hES generated approximately 6.1±0.66 [mean±SD] millionhemangioblasts) under the well-defined and reproducible conditionsdescribed above.

For BL-CFC immunocytochemical analysis, purified BL-CFCs were cytospunonto polylysine treated glass slides and fixed in 4% paraformaldehyde.For examining the expression of most genes, primary antibodies wereincubated at 4° C. overnight, followed by fluorescent dye labeledsecondary antibodies, and finally examined under fluorescent microscope.Normal human BM cells, K562 cells and HUVEC were used as controls.

Immunocytochemical analysis revealed that the hES cell-derived BL-CFCsor BCs expressed GATA-1 and GATA-2 proteins, LMO2 proteins, CXCR-4, TPOand EPO receptors, and readily reacted with antibody specific for CD71,the transferrin receptor (Table 1 and FIG. 16 d-v). The cells expressedlittle or no CD31, CD34 and KDR, or other adhesion molecules. Asdescribed more fully in 11/787,262, the cells are hemangio-colonyforming cells.

Example 15 Expansion of a Distinct Cell Type Non-Engrafting HemangioCell

As detailed above and in Ser. No. 11/787,262, hemangio-colony formingcells were generated following expansion for approximately 4-7 days.Under certain conditions, further culture of EBs beyond 7 days producedlarge numbers of a distinct cell type. As described throughout, thisdistinct progenitor cell type is referred to as a non-engraftinghemangio cell.

EBs were cultured as described above. On day 7 of the expansionprotocol, following formation of grape-like clusters indicative ofhemangio-colony forming cells, 5 ml of BL-medium was added on top of thethese cultures of grape-like clusters of cells. The cultures aresemi-solid and contain 10 mL of methylcellulose medium. Followingaddition of fresh medium, the cells are cultured an additional 3-6 days,for a total of 10-13 days in culture post-EB formation.

The addition of fresh medium greatly enhanced continued cellproliferation and survival during these prolonged culture periods. After10-13 days in culture, cells were purified from the cluster. Similar tohemangio-colony forming cells, these non-engrafting hemangio cellsformed grape-like clusters and were loosely adherent to each other.However, as detailed below, these cells were not identical to thepreviously identified hemangio-colony forming cells.

When the cells were separated from the clusters on day 10, and the yieldof cells compared to the yield of hemangio-colony forming cellsgenerally observed when collected on day 7, we observed a dramaticincrease in the number of cells obtained. Specifically, greater than 5fold more cells were purified on day 10 versus day 7. As such, largerquantities of non-engrafting hemangio cells can be readily produced andused, for example, to produce larger quantities of differentiated celltypes.

The cells identified after 10-13 days of expansion culture are similar,in many respects, to the previously identified hemangio-colony formingcells. For example, the cells are typically loosely adherent to eachother (like hemangio-colony forming cells). Additionally, cellsidentified after 10-13 days of expansion culture differentiated in vitroto produce hematopoietic cell types. Specifically, non-engraftinghemangio cells retain the capacity to form hematopoietic CFUs. Cellswere separated from the grape-like clusters after 10-13 days in cultureand plated in semi-solid methylcellulose medium containing cytokinesthat support growth of hematopoietic CFUs. After 10-12 days in culture,erythrocyte CFUs, granulocyte CFUs, macrophage CFUs, and mixedhematopoietic CFUs were observed, thus demonstrating the potential toproduce hematopoietic cell types.

Despite the similarities between hemangio-colony forming cells and thenon-engrafting hemangio cells described herein, these cells do not havethe same differentiation potential. Without wishing to be bound by anyparticular theory, the non-engrafting hemangio cells may represent adevelopmentally distinct cell type that, in contrast to hemangio-colonyforming cells, are no longer capable of engrafting into the bone marrowupon in vivo delivery to an immunodeficient animal. Specifically, 1-5million human non-engrafting hemangio cells (e.g., cells cultured for10-13 days post-EB formation) were administered to NOD/SCID mice.Examination of 24 mice failed to reveal engraftment of human cells intothe bone marrow or spleen. In contrast, when similar numbers of humanhemangio-colony forming cells (e.g., cells cultured for 6-8 days) wereadministered to NOD/SCID mice, human cells engrafted in the bone marrowof all 12 animals examined.

Other illustrative methods, compositions, preparations, and features ofthe invention are described in the following documents: U.S. applicationSer. No. 11/787,262, filed Apr. 13, 2007, and entitled “Hemangio-ColonyForming Cells.” The teachings of this application are herebyincorporated by reference in their entirety.

It should be noted that Applicants consider all operable combinations ofthe disclosed illustrative embodiments to be patentable subject matterincluding combinations of the subject matter disclosed in U.S.application Ser. No. 11/787,262. For example, the non-engraftinghemangio cells provided herein (i) may have one or more of theproperties of the cells described in U.S. application Ser. No.11/787,262, (ii) may be formulated as compositions, preparations,cryopreserved preparations, or purified or mixed solutions as describedin U.S. application Ser. No. 11/787,262, (iii) may be usedtherapeutically and in blood banking as described in U.S. applicationSer. No. 11/787,262, and (iv) may be used to generate partially andterminally differentiated cell types for in vitro or in vivo use asdescribed in U.S. application Ser. No. 11/787,262. Furthermore, thenon-engrafting hemangio cells can be derived from ES cells, ED cells,pluripotent stem cells (including iPS cells) etc. using any of themethodologies described herein and in U.S. application Ser. No.11/787,262.

Example 16 Efficient Generation of Hemangioblasts from Human iPSCs

Based on the method to efficiently and reproducibly generate largenumbers of hemangioblasts from multiple hESC lines described herein (seealso Lu et al. Nat Methods 2007; 4:501-509; Lu et al. Regen Med 2008;3:693-704), the inventors further used the hemangioblast platform todifferentiate hESCs through hemangioblastic progenitors into erythroidcells on a large scale (approximately 10¹⁰ to 10¹¹ cells/six-well platehESCs), which is over a thousand-fold more efficient than previouslyreported. As discussed supra, the cells possess oxygen-transportingcapacity comparable to normal RBCs and respond to changes in pH (Bohreffect) and 2,3-diphosphoglyerate (DPG) (see also, Lu et al. Blood 2008;112:4475-4484). Importantly, the erythroid cells underwent multiplematuration events in vitro, including a progressive decrease in size andincrease in glycophorin A expression, chromatin and nuclearcondensation, and increased expression of definitive adult β-globinchain. Globin chain specific immunofluorescent analysis showed that thecells (0% at 17 days) increased expression of the adult β-globin chainto 16.37% after 28 days of in vitro culture. This process resulted inthe extrusion of the pycnotic nucleus in 30-60% of the cells generatingRBCs with a diameter of approximately 6-8 μm. The results show that itis feasible to differentiate and mature hESC-derived hemangioblasts intofunctional oxygen-carrying erythrocytes on a large scale.

Human iPSCs share a number of characteristics with hESCs, and representan important new source of stem cells. The identification of an iPSCline with a O(—) genotype would permit the production of ABO and RhDcompatible (and pathogen-free) “universal donor” RBCs, and using apatient's specific iPSC lines would allow the generation of patient'sown platelets in vitro for transfusion. However, little has beenreported about the capacity of iPSCs to undergo directeddifferentiation, especially, toward hemangioblasts. A recent report byChoi et al. (STEM CELLS 2009; 27(3):559-567) describes studies withhuman iPSCs utilizing an OP9 feeder-based culture system that yieldedhematopoietic and endothelial differentiation, demonstrating thepotential of human iPSCs. Similarly, Zhang et al. (Circ Res 2009;104:e30-e41.) reports the derivation of functional cardiomyocytes fromhuman iPSCs, albeit with low efficiency compared to hESCs, using EBmethod. Therefore, efficient generation of hemangioblasts from humaniPSCs is described herein. The inventors describe conditions forefficient generation of hemangioblasts from human iPSCs, using theirexperiences with the hESC system.

Generation of High Quality iPSCs

In several of the inventors' preliminary studies, they are able togenerate hemangioblast colonies from human IMR90 (FIG. 20 c) andAdult4-3 iPSCs (data not shown), using the optimized hESCdifferentiation conditions. Although their efficiency was much lowercompared to hESCs, they clearly demonstrate the hemangioblastdifferentiation potential of human iPSCs. The observed low efficiencymay be due to multiple factors, one of them being the quality of theiPSCs. The inventors observed this to be one of the most importantfactors for high-efficient generation of hemangioblasts. High qualityhESC cultures are composed of colonies with tight borders with minimalsigns of differentiation as seen under microscope, at about 80%confluence, but not touching each other. They grow at a moderate rate:1:3 split passaged hESCs will reach confluence in 3-5 days with positivestaining of pluripotency markers in almost every cell. High qualityhESCs usually generate a high number of EB cells (e.g. 2×10⁶ highquality hESCs will generate=2-3×10⁶ EB cells after 3.5 days). Thecritical steps for obtaining high quality iPSCs include: (1) passagingwith trypsin vs. collagenase: The inventors have demonstrated that hESCscan be routinely passaged by trypsin/EDTA after the initial adaptationfrom mechanically passaged cultures has been performed (Klimanskaya etal. Approaches of derivation and maintenance of human ES cells: Detailedprocedures and alternatives. In: Lanza Rea, ed. Handbook of Stem Cells.Volume 1: Embryonic Stem Cells. New York, USA: Elsevier/Academic Press,2004:437-449.). In the inventors' experience, trypsin works better thanwidely used collagenase IV because it produces smaller cell clumps (2-5cells) and single cells that form more uniformly distributed andsimilarly sized colonies, which will eliminate premature contact betweencolonies and limit spontaneous differentiation, whereas collagenasepassaging results in larger colonies that show more extensivedifferentiation and have to be passed either at a lower splitting ratioor before the desired density of the culture is reached. Overall,trypsin/EDTA passaging allows the ability to scale up the culture 3-4times faster than collagenase and to get a homogenous cell population.These observations may also be valid for human iPSCs. The inventorsexperiments showed that human iPSCs can be adapted to trypsin digestion,and these cells maintain undifferentiated status after more than 20passages; (2) Maintaining with mouse embryonic fibroblasts (MEF feeder)or feeder-free: long term maintenance of hESCs and iPSCs required MEFfeeders. The culturing of hESCs and iPSCs on MEF feeder layers preventscomplete elimination of animal components, and raises concerns for thepotential clinical applications of derivatives generated from hESCs andiPSCs maintained under these conditions. Therefore, the first step hasbeen taken to determine whether hemangioblasts can be generated fromhESCs maintained on Matrigel matrix in mTeSR medium. The inventors havedemonstrated that a significantly higher number (3-fold increase) ofhemangioblasts were generated with feeder-free hESCs as compared tohESCs cultured on MEF feeders when identical numbers of EB cells wereplated (p<0.05) for all three tested hESC lines WA01, MA01 and HuES-3(Lu et al. Regen Med 2008; 3:693-704.). The inventors then initiated theexperiments of culturing human iPSCs in the above feeder-free system,and human iPSCs maintained in feeder-free condition expressed thepluoripotency markers of Nanog, Oct-4, SSEA-4, and Tra-1-60 (FIG. 19).Whether human iPSCs from feeder-free condition will differentiate tohemangioblasts with high efficiency will be tested.

Optimization of Embryoid Body (EB) Formation and Differentiation: Human

iPSCs show poor survival ability after cell dissociation and during EBformation, a phenomenon also observed for hESCs. It has been shown thataddition of a selective Rho-associated kinase (ROCK) inhibitor. Y-27632,to serum-free EB formation medium prevents hESCs from apoptosis,enhances EB formation, and promotes differentiation (Watanabe et al. NatBiotechnol 2007; 25:681-686). The experiments showed that supplement ofY-27632 in the serum-free EB formation and differentiation mediumresulted in better formation of EBs from human iPS(IMR90)-1 cells thancontrol medium: EBs in StemLine II medium plus cytokines only areusually smaller with many dead cells after 24 hrs; whereas EBs in mediumadded with Y-27632 are smooth and large with many fewer dead cellssurrounding them (FIGS. 20 a and 20 b), indicating healthier EBs wereformed. After plating for blast colony formation, cells from EBs treatedwith Y-27632 developed substantial more and healthier blast coloniesthan that derived from EBs without Y-27632 treatment (FIGS. 20 c and 20d), generating >2 fold more hemangioblasts. Previous studies alsosuggest that insulin, a component in almost all cell culture mediaincluding StemLine II medium used in the EB formation system describedherein, is a potent inhibitor of hESC mesoderm differentiation, possiblythrough PI3K/Akt signaling pathway. Inhibition of PI3K/Akt signalingpathway enhanced mesoderm differentiation of hESCs in serum-freeconditions (Freund et al. Stem Cells 2008; 26:724-733.). The resultsshowed that supplementation with a PI3K/Akt signaling pathway inhibitorin EB formation and differentiation medium substantially increased theformation of hemangioblasts from MA09 hESCs. A >2.5 fold increase ofhemangioblasts was obtained from dishes treated with PI3K/Akt inhibitoras compared with dishes from controls. Similarly, supplementation withthe PI3K/Akt signaling pathway inhibitor alone or plus Y-27632 during EBformation also resulted in more and healthier blast colonies fromiPS(IMR90)-1 cells than controls (FIG. 20 e), producing 2.1-fold and 2.6fold more hemangioblasts for PI3K/Art inhibitor treated EBs and PI3K/Artinhibitor plus ROCK inhibitor treated EBs, respectively. Thehemangioblasts were then purified and plated under conditions forhematopoietic or endothelial cell differentiation. As shown in FIG. 20f-20 j, these cells differentiated into both hematopoietic andendothelial cells after replating under appropriate conditions.

Example 17 Directed Differentiation of hESCs into Megakaryocyte andPlatelets

Pluripotent human embryonic stem cells (hESCs) and iPS cells arepotential alternative sources for blood cells used in transfusiontherapies. In addition, directed hESC differentiation into blood canprovide a useful tool to study the ontogeny of hematopoiesis. Efficientand directed differentiation of hESCs into transfusiblemegakaryocytes/platelets is of great clinical significances. However,previously reported methods for generating megakaryocytes and plateletsfrom human ESCs are problematic for potential clinical applications,because 1) the yield of megakaryocytes/platelets from hESCs are too low,2) they require undefined animal stromal cells (e.g., OP9) and 3) thesemethods will be difficult to scale up for massive production (Gaur etal. J Thromb Haemost 2006; 4:436-442; Takayama et al. Blood 2008;111:5298-5306.). A robust model system that can efficiently generatelarge numbers of hemangioblasts (blast cells, BCs) from multiple hESClines using well-defined conditions is described herein (see also Lu etal. Nat Methods 2007; 4:501-509; Lu et al., Regen Med 2008; 3:693-704).These BCs can be further induced to produce functional RBCs in largescale as described herein (see also Lu et al. Blood 2008;112:4475-4484). Since RBCs and megakaryocytes come from commonprogenitors, the explored the possibility of producing megakaryocytesand platelets from our hESC derived hemangioblasts.

Diagram of Culture Methods for Generating Megakaryocytes

Serum free hES cells→Embryoid Body Day 3.5-4→Blast Culture Day6→Megakaryocyte culture Day 7

Three hESC lines are tested so far for MK generation: H1, H7 and HuES-3.Standard protocol was used to generate hemangioblasts (see also Lu etal. Nat Methods 2007; 4:501-509; Lu et al. Regen Med 2008; 3:693-704).Briefly, human ES cells were cultured in serum free media and harvestedfor embryoid body (EB) culture. Day 3 to 4 EB cells were collected andprepared as single cell suspension. 5×10⁵ EB cells were resuspended in 1ml blast growth media for the production of hemangioblasts. Cells fromday 8 hemangioblast culture were harvested for setting up MK culturesuspension in suspension. In summary, the have successfully adapted thehemangioblast model system to efficiently generate megakaryocytes andplatelets from hESCs. Using the improved blast culture method, theinventors can now routinely produce 10 million blast cells from onemillion hESCs after 6 to 8 days of hemangioblast culture (see also Lu etal. Regen Med 2008; 3:693-704). For directed differentiation intomegakaryocyte lineage, these blast cells are harvested and plated inliquid megakaryocyte maturation culture in serum free media supplementedwith defined growth factors including TPO. 1.5 to 2 times increase incell number at the early stage of this culture is usually obtained. Thelimited expansion under the current condition is likely due to the deathof cells committed to other lineages and the initiation of endomitosisof megakaryocytes. By day 4 of liquid maturation culture, greater than90% CD41a+ megakaryocytes can be achieved without the need ofpurification (FIG. 21A). Majority of these CD41a+ megakaryocytes areco-expressing CD42b, an additional marker for megakaryocytes. As aresult, 8 to 9 million CD41+ megakaryocytes can be produced from onemillion hESCs in 14 to 15 days. In comparison, the most recent articleby Takayama et al. reported the generation of 2 million CD41a+megakaryocytes (50% of total population) from one million hESCs using aco-culture system with OP9 stromal cells and fetal bovine serum(Takayama et al. Blood 2008; 111:5298-5306). Clearly, hemangioblastsystem described herein represents a significant improvement for invitro generation of megakaryocytes from hESCs.

In addition to cell surface markers, Giemsa staining shows thatmegakaryocytes in maturation culture increase in cell size, undergoendomitosis and become polyploid (FIG. 21C). Furthermore, specificimmunostaining of von Willebrand Factor (VWF) in cellular granulesindicates that the cytoplasmic maturation process occurs in these cells(FIG. 21D). By day 6 of liquid maturation culture, greater than 50% ofCD41a+ cells show >4 n DNA content by FACS analysis (FIG. 21B).Importantly, these in vitro derived megakaryocytes undergo terminaldifferentiation by showing proplatelet formation, an essential steptowards thrombopoiesis (FIG. 21E). With the current conditions describedherein, proplatelet forming cells are observed as early as day 3 inliquid culture and usually reach to a peak of 2-3% of viable cells byday 7 to 8.

After the removal of cells by centrifugation, the supernatant ofmegakaryocyte maturation culture was examined for platelet generation.Indeed, CD41a+ particles are detected and their forward and side scattercharacteristics are very similar to human peripheral blood plateletscontrols used in our FACS analysis (FIG. 22).

REFERENCES

-   1. Wagner R C: Endothelial cell embryology and growth. Adv.    Microcirc. 9, 45-75 (1980).-   2. Lu S J, Feng Q, Caballero S et al: Generation of functional    hemangioblasts from human embryonic stem cells. Nat. Methods 4(6),    501-509 (2007).-   3. Wang L, Li L, Shojaei F et al: Endothelial and hematopoietic cell    fate of human embryonic stem cells originates from primitive    endothelium with hemangioblastic properties. Immunity. 21(1), 31-41    (2004).-   4. Zambidis E T, Peault B, Park T S, Bunz F, Civin C I:    Hematopoietic differentiation of human embryonic stem cells    progresses through sequential hematoendothelial, primitive, and    definitive stages resembling human yolk sac development. Blood    106(3), 860-870 (2005).-   5. Kennedy M, D'Souza S L, Lynch-Kattman M, Schwantz S, Keller G:    Development of the hemangioblast defines the onset of hematopoiesis    in human ES cell differentiation cultures. Blood 109(7), 2679-2687    (2007).-   6. Klimanskaya I, McMahon J. Approaches of derivation and    maintenance of human ES cells: Detailed procedures and alternatives.    In: Handbook of Stem Cells. Volume 1: Embryonic Stem Cells. Lanza    Rea (Eds.). Elsevier/Academic Press, 2004)-   7. Ludwig T E, Bergendahl V, Levenstein M E, Yu J, Probasco M D,    Thomson J A: Feeder-independent culture of human embryonic stem    cells. Nat. Methods 3(8), 637-646 (2006).-   8. Ludwig T E, Levenstein M E, Jones J M et al: Derivation of human    embryonic stem cells in defined conditions. Nat. Biotechnol. 24(2),    185-187 (2006).-   9. Livak K J, Schmittgen T D: Analysis of relative gene expression    data using real-time quantitative PCR and the 2(−Delta Delta C(T))    Method. Methods 25(4), 402-408 (2001).-   10. Lu S J, Feng Q, Ivanova Y et al: Recombinant HoxB4 Fusion    Proteins Enhance Hematopoietic Differentiation of Human Embryonic    Stem Cells. Stem Cells Dev. 16(4), 547-560 (2007).-   11. Soker S, Takashima S, Miao H Q, Neufeld G, Klagsbrun M:    Neuropilin-1 is expressed by endothelial and tumor cells as an    isoform-specific receptor for vascular endothelial growth factor.    Cell 92(6), 735-745 (1998).-   12. Faloon P, Arentson E, Kazarov A et al: Basic fibroblast growth    factor positively regulates hematopoietic development. Development    127(9), 1931-1941 (2000).-   13. Pearson S, Sroczynska P, Lacaud G, Kouskoff V: The stepwise    specification of embryonic stem cells to hematopoietic fate is    driven by sequential exposure to Bmp4, activin A, bFGF and VEGF.    Development 135(8), 1525-1535 (2008).-   14. Pick M, Azzola L, Mossman A, Stanley E G, Elefanty A G:    Differentiation of human embryonic stem cells in serum-free medium    reveals distinct roles for bone morphogenetic protein 4, vascular    endothelial growth factor, stem cell factor, and fibroblast growth    factor 2 in hematopoiesis. Stem Cells 25(9), 2206-2214 (2007).-   15. Martin M J, Muotri A, Gage F, Varki A: Human embryonic stem    cells express an immunogenic nonhuman sialic acid. Nat. Med. 11(2),    228-232 (2005).-   16. Huber T L, Zhou Y, Mead P E, Zon L I: Cooperative effects of    growth factors involved in the induction of hematopoietic mesoderm.    Blood 92(11), 4128-4137 (1998).-   17. Winnier G, Blessing M, Labosky P A, Hogan B L: Bone    morphogenetic protein-4 is required for mesoderm formation and    patterning in the mouse. Genes Dev. 9(17), 2105-2116 (1995).-   18. Johansson B M, Wiles M V: Evidence for involvement of activin A    and bone morphogenetic protein 4 in mammalian mesoderm and    hematopoietic development. Mol. Cell Biol. 15(1), 141-151 (1995).-   19. Wiles M V, Johansson B M: Embryonic stem cell development in a    chemically defined medium. Exp. Cell Res. 247(1), 241-248 (1999).-   20. Nakayama N, Lee J, Chiu L: Vascular endothelial growth factor    synergistically enhances bone morphogenetic protein-4-dependent    lymphohematopoietic cell generation from embryonic stem cells in    vitro. Blood 95(7), 2275-2283 (2000).-   21. Li F, Lu S-J, Vida L, Thomson J A, Honig G R: Bone morphogenetic    protein-4 induces efficient hematopoietic differentiation of rhesus    monkey embryonic stem cells in vitro. Blood 98(2), 335-342 (2001).-   22. Tian X, Morris J K, Linehan J L, Kaufman D S: Cytokine    requirements differ for stroma and embryoid body-mediated    hematopoiesis from human embryonic stem cells. Exp. Hematol. 32(10),    1000-1009 (2004).-   23. Chadwick K, Wang L, Li L et al: Cytokines and BMP-4 promote    hematopoietic differentiation of human embryonic stem cells. Blood    2003 102(3), 906-915 (2003).-   24. Cerdan C, Rouleau A, Bhatia M: VEGF-A165 augments erythropoietic    development from human embryonic stem cells. Blood 103(7), 2504-2512    (2004).-   25. Park C, Afrikanova I, Chung Y S et al: A hierarchical order of    factors in the generation of FLK- and SCL-expressing hematopoietic    and endothelial progenitors from embryonic stem cells. Development    131(11), 2749-2762 (2004).-   26. Xu C, Rosier E, Jiang J et al: Basic fibroblast growth factor    supports undifferentiated human embryonic stem cell growth without    conditioned medium. Stem Cells 23(3), 315-323 (2005).-   27. Xu R H, Peck R M, Li D S, Feng X, Ludwig T, Thomson J A: Basic    FGF and suppression of BMP signaling sustain undifferentiated    proliferation of human ES cells. Nat. Methods 2(3), 185-190 (2005).-   28. Levenstein M E, Ludwig T E, Xu R H et al: Basic fibroblast    growth factor support of human embryonic stem cell self-renewal.    Stem Cells 24(3), 568-574 (2006).-   29. Yeoh J S, van O R, Weersing E et al: Fibroblast growth factor-1    and -2 preserve long-term repopulating ability of hematopoietic stem    cells in serum-free cultures. Stem Cells 24(6), 1564-1572 (2006).-   30. Dell'Era P, Ronca R, Coco L, Nicoli S, Metra M, Presta M:    Fibroblast growth factor receptor-1 is essential for in vitro    cardiomyocyte development. Circ. Res. 93(5), 414-420 (2003).-   31. de H G, Weersing E, Dontje B et al: In vitro generation of    long-term repopulating hematopoietic stem cells by fibroblast growth    factor-1. Dev. Cell 4(2), 241-251 (2003).-   32. Ginis I, Luo Y, Miura T et al: Differences between human and    mouse embryonic stem cells. Dev. Biol. 269(2), 360-380 (2004).-   33. D'Amour K A, Agulnick A D, Eliazer S, Kelly O G, Kroon E, Baetge    E E: Efficient differentiation of human embryonic stem cells to    definitive endoderm. Nat. Biotechnol. 23(12), 1534-1541 (2005).

Various embodiments of the invention are described above in the DetailedDescription. While these descriptions directly describe the aboveembodiments, it is understood that those skilled in the art may conceivemodifications and/or variations to the specific embodiments shown anddescribed herein. Any such modifications or variations that fall withinthe purview of this description are intended to be included therein aswell. Unless specifically noted, it is the intention of the inventorsthat the words and phrases in the specification and claims be given theordinary and accustomed meanings to those of ordinary skill in theapplicable art(s).

The foregoing description of various embodiments of the invention knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the invention to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe invention and its practical application and to enable others skilledin the art to utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.Therefore, it is intended that the invention not be limited to theparticular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this invention. It will be understood by those within the art that,in general, terms used herein are generally intended as “open” terms(e.g., the term “including” should be interpreted as “including but notlimited to,” the term “having” should be interpreted as “having atleast,” the term “includes” should be interpreted as “includes but isnot limited to,” etc.).

1. A method of producing a pluripotent stem cell-derived enucleatederythroid cell, comprising: providing a pluripotent stem cell; anddifferentiating said pluripotent stem cell into an enucleated erythroidcell by culturing said pluripotent stem cell with OP9 mouse stromalcells or human mesenchymal stem cells (MSCs).
 2. The method of claim 1,wherein said pluripotent stem cell is an embryonic stem cell orembryo-derived cell.
 3. The method of claim 1, wherein said pluripotentstem cell is an induced pluripotent stem cell.
 4. The method of claim 1,wherein said pluripotent stem cell is a human cell.
 5. The method ofclaim 1, wherein said pluripotent stem cell is genetically manipulatedprior to differentiation.
 6. The method of claim 1, whereindifferentiating said pluripotent stem cell into an enucleated erythroidcell comprises differentiating said pluripotent stem cell into ahemangioblast, non-engrafting hemangio cell, or blast cell.
 7. Themethod of claim 6, wherein said hemangioblast, non-engrafting hemangiocell, or blast cell is expanded prior to being differentiated into saidenucleated erythroid cell.
 8. The method of claim 7, wherein saidhemangioblasts, non-engrafting hemangio cells, or blast cells areexpanded in Stemline II medium with Epo, IL-3, and SCF.
 9. The method ofclaim 6, wherein said pluripotent stem cell is a human pluripotent stemcell and differentiating said human pluripotent stem cell into saidhemangioblast is done in vitro by a method comprising: (a) culturing acell culture comprising human pluripotent stem cell in serum-free mediain the presence of at least one growth factor in an amount sufficient toinduce the differentiation of said human pluripotent stem cell intoembryoid bodies; and (b) adding at least two growth factors to saidculture comprising embryoid bodies and continuing to culture saidculture in serum-free media, wherein said growth factor is in an amountsufficient to expand said human hemangioblast in said embryoid bodiesculture, wherein said human pluripotent stem cells, embryoid bodies andhemangioblasts are grown in serum-free media throughout steps (a) and(b) of said method, and wherein said at least two growth factors in step(b) comprise BMP4 and VEGF.
 10. The method of claim 9, whereindifferentiating said human pluripotent stem cell into said hemangioblastfurther comprises: (c) disaggregating said embryoid bodies into singlecells; and (d) adding at least one growth factor to said culturecomprising said single cells and continuing to culture said culture inserum-free media, wherein said growth factor is in an amount sufficientto expand human hemangioblasts in said culture comprising said singlecells, and wherein said human pluripotent stem cells, embryoid bodiesand hemangio-colony foaming cells are grown in serum-free mediathroughout steps (a)-(d) of said method. 11-18. (canceled)
 19. Themethod of claim 6, wherein said pluripotent stem cell is a humanpluripotent stem cell and differentiating said human pluripotent stemcell into said non-engrafting hemangio cell is done in vitro by a methodcomprising: (a) culturing a cell culture comprising said humanpluripotent stem cell in serum-free media in the presence of at leastone growth factor in an amount sufficient to induce the differentiationof said human pluripotent stem cell into embryoid bodies; and (b) addingat least one growth factor to said culture comprising embryoid bodiesand continuing to culture said culture in serum-free media, wherein saidgrowth factor is in an amount sufficient to expand said humannon-engrafting hemangio cell in said embryoid bodies culture, whereinsaid embryoid bodies are cultured for 10-13 days, and wherein said humanpluripotent stem cell, embryoid bodies and non-engrafting hemangio cellsare grown in serum-free media throughout steps (a) and (b) of saidmethod.
 20. The method of claim 19, wherein differentiating saidpluripotent stem cell into said non-engrafting hemangio cell furthercomprises: (c) disaggregating said embryoid bodies into single cells;and (d) adding at least one growth factor to said culture comprisingsaid single cells and continuing to culture said culture in serum-freemedia, wherein said growth factor is in an amount sufficient to expandsaid human non-engrafting hemangio cell in said culture comprising saidsingle cells, wherein said embryo-derived cells, embryoid bodies andnon-engrafting hemangio cells are grown in serum-free media throughoutsteps (a)-(d) of said method. 21-28. (canceled)
 29. The method of claim1, wherein differentiating said pluripotent stem cell into saidenucleated erythroid cell further comprises culturing said pluripotentstem cell in the culture medium comprising EPO.
 30. The method of claim29, wherein differentiating said pluripotent stem cell into saidenucleated erythroid cell further comprises: culturing said pluripotentstem cell in a culture medium comprising a supplement selected from thegroup consisting of inositol, folic acid, monothioglycerol, transferrin,insulin, ferrous nitrate, ferrous sulfate, BSA, L-glutamine,penicillin-streptomycin and combinations thereof; and culturing saidpluripotent stem cell in said culture medium wherein said culture mediumfurther comprises an agent selected from the group consisting ofhydrocortisone, SCF, IL3, Epo and combinations thereof.
 31. Anenucleated erythroid cell produced by the method of claim
 1. 32. Amethod of producing a pluripotent stem cell-derived erythroid cell,comprising: providing a pluripotent stem cell; and differentiating saidpluripotent stem cell into an erythroid cell by culturing saidpluripotent stem cell in a medium comprising EPO.
 33. The method ofclaim 32, wherein said pluripotent stem cell is an embryonic stem cellor embryo-derived cell.
 34. The method of claim 32, wherein saidpluripotent stem cell is an induced pluripotent stem cell.
 35. Themethod of claim 32, wherein said pluripotent stem cell is a human cell.36. The method of claim 32, wherein said pluripotent stem cell isgenetically manipulated prior to differentiation.
 37. The method ofclaim 32, wherein differentiating said pluripotent stem cell into anerythroid cell comprises differentiating said pluripotent stem cell intoa hemangioblast, non-engrafting hemangio cell, or blast cell.
 38. Themethod of claim 37, wherein said hemangioblast, non-engrafting hemangiocell, or blast cell is expanded prior being differentiated into saiderythroid cell.
 39. The method of claim 38, wherein said hemangioblasts,non-engrafting hemangio cells, or blast cells are expanded in StemlineII medium with Epo, IL-3, and SCF.
 40. The method of claim 32, whereinsaid pluripotent stem cell is a human pluripotent stem cell anddifferentiating said human pluripotent stem cell into said hemangioblastis done in vitro by a method comprising: (a) culturing a cell culturecomprising said human pluripotent stem cell in serum-free media in thepresence of at least one growth factor in an amount sufficient to inducethe differentiation of said human pluripotent stem cell into embryoidbodies; and (b) adding at least two growth factors to said culturecomprising embryoid bodies and continuing to culture said culture inserum-free media, wherein said growth factor is in an amount sufficientto expand said human hemangioblast in said embryoid bodies culture,wherein said human pluripotent stem cells, embryoid bodies andhemangioblasts are grown in serum-free media throughout steps (a) and(b) of said method, and wherein said at least two growth factors in step(b) comprise BMP4 and VEGF.
 41. The method of claim 40, whereindifferentiating said human pluripotent stem cell into said hemangioblastfurther comprises: (c) disaggregating said embryoid bodies into singlecells; and (d) adding at least one growth factor to said culturecomprising said single cells and continuing to culture said culture inserum-free media, wherein said growth factor is in an amount sufficientto expand human hemangioblasts in said culture comprising said singlecells, and wherein said pluripotent stem cells, embryoid bodies andhemangio-colony forming cells are grown in serum-free media throughoutsteps (a)-(d) of said method. 42-49. (canceled)
 50. The method of claim32, wherein said pluripotent stem cell is a human pluripotent stem celland differentiating said pluripotent stem cell into said non-engraftinghemangio cell is done in vitro by a method comprising: (a) culturing acell culture comprising human pluripotent stem cell in serum-free mediain the presence of at least one growth factor in an amount sufficient toinduce the differentiation of said human pluripotent stem cell intoembryoid bodies; and (b) adding at least one growth factor to saidculture comprising embryoid bodies and continuing to culture saidculture in serum-free media, wherein said growth factor is in an amountsufficient to expand said human non-engrafting hemangio cells in saidembryoid bodies culture, wherein said embryoid bodies are cultured for10-13 days, and wherein said human pluripotent stem cell, embryoidbodies and non-engrafting hemangio cells are grown in serum-free mediathroughout steps (a) and (b) of said method.
 51. The method of claim 50,wherein differentiating said pluripotent stem cell into saidnon-engrafting hemangio cell further comprises (c) disaggregating saidembryoid bodies into single cells; and (d) adding at least one growthfactor to said culture comprising said single cells and continuing toculture said culture in serum-free media, wherein said growth factor isin an amount sufficient to expand human non-engrafting hemangio cells insaid culture comprising said single cells, wherein said humanpluripotent stem cell, embryoid bodies and non-engrafting hemangio cellsare gown in scrum-free media throughout steps (a)-(d) of said method.52-57. (canceled)
 58. An erythroid cell produced by the method of claim32.
 59. A method of producing a megakaryocyte or a platelet, comprising:providing a pluripotent stem cell; differentiating said pluripotent stemcell into a hemangioblast, non-engrafting hemangio cell, or blast cell;and differentiating said hemangioblast, non-engrafting hemangio cell, orblast cell into said megakaryocyte or said platelet by culturing inmegakaryocyte (MK) culture medium comprising TPO.
 60. The method ofclaim 59, wherein said pluripotent stem cell is an embryonic stem cellor embryo-derived cell.
 61. The method of claim 59, wherein saidpluripotent stem cell is an induced pluripotent stem cell.
 62. Themethod of claim 59, wherein said pluripotent stern cell is a human cell.63. The method of claim 59, wherein said pluripotent stem cell isgenetically manipulated prior to differentiation.
 64. The method ofclaim 59, wherein said hemangioblast, non-engrafting hemangio cell, orblast cell is expanded prior to being differentiated into saidmegakaryocyte or said platelet.
 65. The method of claim 64, wherein saidhemangioblasts, non-engrafting hemangio cells, or blast cells areexpanded in Stemline II medium with Epo, IL-3, and SCF.
 66. The methodof claim 59, wherein said pluripotent stem cell is a human pluripotentstem cell and differentiating said human pluripotent stem cell into saidhemangioblast is done in vitro by a method comprising: (a) culturing acell culture comprising human pluripotent stem cell in serum-free mediain the presence of at least one growth factor in an amount sufficient toinduce the differentiation of said human pluripotent stem cell intoembryoid bodies; and (b) adding at least two growth factors to saidculture comprising embryoid bodies and continuing to culture saidculture in serum-free media, wherein said growth factor is in an amountsufficient to expand said human hemangioblast in said embryoid bodiesculture, wherein said human pluripotent stem cells, embryoid bodies andhemangioblasts are grown in serum-free media throughout steps (a) and(b) of said method, and wherein said at least two growth factors in step(b) comprise BMP4 and VEGF.
 67. The method of claim 66, whereindifferentiating said human pluripotent stem cell into said hemangioblastfurther comprises: (c) disaggregating said embryoid bodies into singlecells; and (d) adding at least one growth factor to said culturecomprising said single cells and continuing to culture said culture inserum-free media, wherein said growth factor is in an amount sufficientto expand human hemangioblasts in said culture comprising said singlecells, and wherein said human pluripotent stem cells, embryoid bodiesand hemangio-colony forming cells are grown in serum-free mediathroughout steps (a)-(d) of said method. 68-73. (canceled)
 74. Themethod of claim 59, wherein differentiating said hemangioblast,non-engrafting hemangio cell, or blast cell into said megakaryocyte orsaid platelet is done after about 6 to 8 days of hemangioblast,non-engrafting hemangio cell, or blast cell culture.
 75. The method ofclaim 59, wherein said pluripotent stem cell is a human pluripotent stemcell and differentiating said human pluripotent stem cell into saidnon-engrafting hemangio cell is done in vitro by a method comprising:(a) culturing a cell culture comprising said human pluripotent stem cellin serum-free media in the presence of at least one growth factor in anamount sufficient to induce the differentiation of said humanpluripotent stem cell into embryoid bodies; and (b) adding at least onegrowth factor to said culture comprising embryoid bodies and continuingto culture said culture in serum-free media, wherein said growth factoris in an amount sufficient to expand said human non-engrafting hemangiocell in said embryoid bodies culture, wherein said embryoid bodies arecultured for 10-13 days, and wherein said human pluripotent stem cell,embryoid bodies and non-engrafting hemangio cells are grown inserum-free media throughout steps (a) and (b) of said method.
 76. Themethod of claim 75, wherein differentiating said pluripotent stem cellinto said non-engrafting hemangio cell further comprises: (c)disaggregating said embryoid bodies into single cells; and (d) adding atleast one growth factor to said culture comprising said single cells andcontinuing to culture said culture in serum-free media, wherein saidgrowth factor is in an amount sufficient to expand said humannon-engrafting hemangio cell in said culture comprising said singlecells, wherein said embryo-derived cells, embryoid bodies andnon-engrafting hemangio cells are grown in serum-free media throughoutsteps (a)-(d) of said method. 77-82. (canceled)
 83. A megakaryocyte or aplatelet produced by the method of claim 59.